Recombinant microorganisms expressing an oligosaccharide receptor mimic

ABSTRACT

Chimeric carbohydrates produced by recombinant microorganism carrying exogenous glycosyltransferases act with or without exogenous enzymes required for synthesis or nucleotide synthesis precursors. These recombinant microorganism can be used for competitively inhibiting the binding of toxins or adhesins to receptors of mucosal surfaces, especially gastrointestinal surface. In particular chimeric sugar moieties have been made for lipopolysaccharides, in recombinant microorganism that present multiple copies of the oligosaccharides. The oligosaccharide moieties so presented act as receptor mimic for toxins and adhesins. A number have been synthesized and have been shown to confer protection against attack by pathogenic organisms or their products in vitro and in vivo.

FIELD OF THE INVENTION

This invention relates to recombinant microorganisms (i.e., bacteria,yeast, and fungi) that display an oligosaccharide-comprising bindingmoiety that can compete with a ligand for binding to a receptor for theligand, and use of the microorganisms to deliver the oligosaccharide toa human or other animal. The microoganism can be used for adsorbingtoxins or pathogenic microorganisms from a particular environment.

BACKGROUND OF THE INVENTION

Surfaces of all cells express a complexity of oligosaccharides thatprovide a number of functions. A primary one of these functions isdetermining on their own or together with other molecules, interactionswith other cells or molecules. The nature, linkage and conformation ofsugar residues of the oligosaccharides, and in particular residues at orclose to the non-reducing terminus of an oligosaccharide, determineswhether the oligosaccharide will or will not participate in a particularreceptor-ligand interaction.

The susceptibility of an animal to infection and the eventualphysiological site affected by an infection is to a large extentdetermined by the expression on the cell surface of such oligosaccharidereceptors. In the case of enteric infections, one primary prerequisitefor pathogenesis is that the microorganism persists in the intestinallumen of the host. Generally this requires some form of adherence to thelumeinal epithelium (forming the mucosal surface of the gut), otherwisethe micro-organism is flushed from the gut. Additionally for a toxigenicorganism, the toxin also needs to bind to the luminal epithelium and,for some toxins, needs to be absorbed systemically to be effective,otherwise, it too would be flushed from the gut.

Certain surface structures of pathogenic and other bacteria known asadhesins mediate adherence to luminal epithelial cells. A number ofadhesins are known and organisms without adhesins are generally of lowvirulence. Adhesins are proteinaceous factors which promote theadherence of bacteria and viruses to cells of their hosts. Adhesins canbe either fimbrial or filamentous in structure or they may be afimbrial.Adhesins associated with fimbriae may be associated with accessoryproteins such as tip proteins at an extreme end of the fimbrialstructure and such tip proteins can be regarded as lectins. The receptoron the host cell has in some cases been determined and shown to be acarbohydrate such as an oligosaccharide associated with a glycolipid ora glycoprotein.

The best characterised system from a molecular and biological viewpoint,is the P-fimbriae (also called pili) produced by uropathogenicEscherichia coli. This tip adhesin binds the glycolipid Gb₃(globotriaosyl ceramide—see below) and the fimbrial subunits can bepurified by affinity chromatography using a Gb₃ mimic. Another wellcharacterised group of adhesins are those associated withenterotoxigenic E. coli (ETEC) strains which infect pigs to causescours. These are termed the K88 type and a number of variants areknown, being K88ab, K88ac and K88ad. The adhesins associated with thesefimbriae have been shown to have different receptor requirements, whichincludes the presence of both glycolipid and protein receptors. Thecarbohydrate requirement has been characterised in at least some ofthese, for example K88ad uses carbohydrates of the lactoneotetraoseseries of glycolipids.

A number of toxins have been identified and include, Shiga toxins (alsoreferred to as Shiga-like toxins and verotoxins), toxins produced byvarious species of Clostridia, including tetanus toxin, botulinum toxin,and C. diffcile toxins A and B, Staphylococcal enterotoxins, Escherichiacoil heat labile and heat stable enterotoxins and cholera toxin. Withouttoxin activity the majority of otherwise enterotoxigenic bacteria wouldbe less capable of causing disease.

The receptors for the majority of adhesins and toxins identified to dateare carbohydrate in nature. For example, the glycolipid globotriaosylceramide (Gb₃) which has the structure Galα[1→4]Galβ[1→4]Glc-ceramide,is the preferred receptor for most members of the Shiga toxin family.Similarly, the ganglioside G_(M1) is the receptor for cholera toxin andE. coli heat labile enterotoxin type I. C. difficile toxin A binds toseveral host receptors, all of which have in common a Galβ[1→4]GlcNAcmoiety. The neurotoxin produced by C. botulinum is also believed to bespecific for a sialic acid containing glycoprotein or glycolipid presenton neurons. The terminal Galα[1→4]Galβ moiety present on Gb₃ is thereceptor for P pili, the major adhesin of uropathogenic E. coli strains.Similarly, asialo-G_(M1) is the receptor for adhesive pili (CFAs) ofsome enterotoxigenic E. coli strains. The sialated gangliosidesNeuGc-GM₃ and NeuNAc-GM₃ have also been identified as the target cellreceptors for porcine rotavirus strains, and it is presumed thatrotavirus strains causing disease in humans also bind specificoligosaccharide moieties present on cell surface glycolipids.

The elucidation of the nature of oligosaccharides acting as receptorsfor particular toxins and pathogenic microorganisms has opened up apromising avenue in the diagnosis and potential treatment or preventionof diseases caused by these agents. The use of the sugar residuesforming receptors for toxins or adhesins has been proposed as a means ofspecifically identifying the toxins or bacteria involved in aninfection. For example, the ganglioside receptor G_(M1) is used as aspecific capturing agent in ELISA assays for the presence of choleratoxin.

It has also been proposed to use synthetically prepared oligosaccharidesas a means of adsorbing toxins or the like from samples. Examples ofproposed uses of receptors for adsorbing toxins or pathogenic organismsout of a sample include Krivan et al in U.S. Pat. No. 5,696,000 whichdiscusses the pharmaceutical use of certain tetra- and tri- saccharidereceptors coupled to a carrier such as a liposome to inhibit theadherence of micro-organisms to susceptible cells. A similar use fortoxins such as Shiga toxin, can be seen in U.S. Pat. No. 5,849,714 toRafter et al which discloses the use of a synthetic construct of sugarresidues making up the globotriose receptor, coupled by a linker to aninert support for use in treatment of bacterial dysentery.

A problem arises, however, in the synthesis and delivery of thesecompounds, because oligosaccharides are difficult or expensive tosynthesise chemically, the conformation may not be appropriate, and theoligosaccharide may preferably need to be presented in an immobilized(non-diffusible) form. Thus, there is a need to provide a mechanism fordelivery and presentation of the oligosaccharide moiety in anappropriate conformation in the environment where the toxin orpathogenic organism is to be adsorbed (for example the gastrointestinaltract).

SUMMARY OF THE INVENTION

The invention provides, in a first embodiment, a recombinantmicroorganism that displays on its surface a binding moiety that, whenadministered to an animal, competes with a ligand for binding to areceptor for the ligand. The binding moiety includes an oligosaccharidethat is composed of at least one sugar residue that is attached to anacceptor moiety by a glycosyltransferase that is encoded by an exogenousnucleic acid which is present in the microorganism. The oligosaccharidecan further include at least a second sugar residue that is attached tothe acceptor moiety by at least a second glycosyltransferase. One ormore of the additional glycosyltransferases can also be encoded by oneor more exogenous nucleic acids that are present in the microorganism.

The receptor is typically present on a surface of a cell. Cells ofinterest include, for example epithelial or endothelial cells, inparticular those that are present in an animal mucosal membrane.

The binding moiety is, in some embodiments, a mimic of a receptor for atoxin or adhesin of a pathogenic organism. Examples of toxins include,but are not limited to, enterotoxins, including shiga toxins,clostridial toxins, cholera toxins, E. coli enterotoxins, andStaphylococcal enterotoxins. In other embodiments, the binding moiety isa mimic of an adhesin receptor. Adhesins of interest include, but arenot limited to, a CFA adhesin of an enterotoxigenic E coli., E. coli CS3pili, K88ad fimbriae, an adhesin of Entamoeba histolyticum, and anadhesin of a virus.

In some embodiments, the binding moiety competes with a pathogenicorganism for binding to a corresponding receptor on an animal epithelialor endothelial cell. Pathogenic organisms of interest include, forexample, Staphylococcus pneumonia, H. influenza, H. parainfluenza,Chlamydia trachomatis, Acanthamoeba, Candida albicans, Helicobacterpylori and Pseudomonas spp.

In other embodiments, the binding moiety is a mimic of a receptor for acell or molecule involved in inflammation. For example, the bindingmoiety can include a 3′-sialoside or a 6′-sialoside. Sialyl Lewis^(x)and sialyl Lewis^(a) are other examples of oligosaccharide structuresthat can function as a receptor mimic. Microorganisms that display thesemolecules can compete with leukocytes, for example, for binding toendothelial cells in the vasculature or other tissues and thus inhibitinflammation.

The invention also provides a recombinant microorganism expressing oneor more exogenous sugar transferases, or one or more exogenousnucleotide sugar precursor synthesizing enzymes, said microorganism alsoexpressing an acceptor molecule, said one or more exogenous sugartransferases being specific for the transfer of one or more sugarresidues represented progressively from a non reducing terminal end of areceptor of either a toxin or an adhesin of a pathogenic organism, theexogenous sugar transferases progressively transferring said one or moresugar resides onto the acceptor molecule to thereby form a chimericcarbohydrate molecule with an exposed receptor mimic, said sugarprecursor enzymes forming nucleotide precursors that are transferred tosaid acceptor molecule to make up said chimeric carbohydrate, saidexposed receptor mimic capable of binding the toxin or the adhesin.

Also provided by the invention are preparations for administration to amucosal surface. The preparations include a delivery microorganism or apartially or fully purified non-toxic preparation of a carbohydratemolecule therefrom. At least a part of the carbohydrate moleculegenerally acts as an exposed receptor mimic. For example, the receptormimic can be capable of binding a toxin or an adhesin of a pathogen thatnormally binds to the mucosal surface. The receptor mimic is generallycarried in a pharmaceutically acceptable excipient.

The invention also provides methods of administering a receptor mimic toa mucosal surface of a mammal. The methods involve the administration ofa quantity of a delivery microorganism, or parts thereof, the deliverymicroorganism exhibiting one or more sugars in a configuration to forman exposed receptor mimic, the receptor mimic being a mimic of areceptor of a pathogen, said quantity being sufficient to reduceadherence of the pathogen or a toxin produced by the pathogen to themucosal surface.

In another embodiment, the invention provides methods of testing for thepresence of a toxin or a pathogenic microorganism in a sample. Thesemethods involve, for example, contacting a sample with the purifiedcarbohydrate as described above, with either the purified carbohydrateor the sample being immobilized. Unbound purified carbohydrate or toxinor pathogenic microorganism is washed off, and a detection means isadded to detect bound purified carbohydrate and the toxin or pathogenicmicroorganism.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a plot showing the protective efficacy offormaldehyde-killed-receptor mimic bacteria. Groups of sixstreptomycin-treated mice were challenged with 97MW1, and then treatedorally twice or three times daily with E. coli CWG308 orCWG308:pJCP-Gb₃. The survival time of each mouse is shown.

FIG. 2 shows representations of histological confirmation ofSTEC-induced renal injury. Kidneys were removed from an uninfectedcontrol mouse (A), a CWG308-treated mouse which died four days afterchallenge with 97MW1 (B), and two different CWG308:pJCP-Gb₃ treated micewhich were alive and well 12 days after challenge with 97MW1 (C and D).Kidneys were fixed, sectioned and stained with H&E as described thematerials and methods of example 2.

FIG. 3 is a plot showing the effect of delayed therapy withCWG308:pJCP-Gb₃ on survival of mice challenged with 97MW1. Groups offour mice were challenged with 97MW1 and treatment with CWG308:pJCP-Gb₃was commenced either immediately, after a delay of 8, 16, 24 or 48hours, or not at all. Survival time of each mouse is indicated.

FIG. 4 is a plot showing the effect of delayed therapy withCWG308:pJCP-Gb₃ on survival of mice challenged with 98NK2. Groups ofeight mice were challenged with 98K2 and treatment with CWG308:pJCP-Gb₃was commenced either immediately, after a delay of 8, 16, 24 or 48hours, or not at all. Survival time of each mouse is indicated.

FIG. 5 shows representations of silver stained SDS-PAGE analysis of LPSpurified from E. coli CW308 derivatives. Lane 1, CWG308; lane 2,CWG308:pJCP-Gb₃; lane 2, CWG308:pJCP-lgtCDE; lanes 4 and 5CWG308:pJCP-lgtCDE/gne (two separate preparations); lane 6CWG308:pJCP-lgtCDE/wbnF. Each lane contains approximately 3 μg of LPS.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The following abbreviations are used herein:

Ara=arabinosyl;

Fru=fructosyl;

Fuc=fucosyl;

Gal=galactosyl;

GalNAc=N-acetylgalactosaminyl;

GIc=glucosyl;

GlcNAc=N-acetylglucosaniinyl;

Man=mannosyl; and

NeuAc or NeuNAc=sialyl (N-acetylneuraminyl).

Typically, sialic acid is 5-N-acetylneuraminic acid, (NeuAc) or5-N-glycolylneuraminic acid (NeuGc). Other sialic acids may be used intheir place, however. For a review of different forms of sialic acidsuitable in the present invention see, Schauer, Methods in Enzymologly,50: 64-89 (1987), and Schaur, Advances in Carbohydrate Chemistry andBiochemistry, 40: 131-234.

Donor substrates for glycosyltransferases are activated nucleotidesugars. Such activated sugars generally consist of uridine and guanosinediphosphate, and cytidine monophosphate, derivatives of the sugars inwhich the nucleoside diphosphate or monophosphate serves as a leavinggroup. Bacterial, plant, and fungal systems can sometimes use otheractivated nucleotide sugars.

Oligosaccharides are considered to have a reducing end and anon-reducing end, whether or not the saccharide at the reducing end isin fact a reducing sugar. In accordance with accepted nomenclature,oligosaccharides are depicted herein with the non-reducing end on theleft and the reducing end on the right. All oligosaccharides describedherein are described with the name or abbreviation for the non-reducingsaccharide (e.g., Gal), followed by the configuration of the glycosidicbond (α or β), the ring bond, the ring position of the reducingsaccharide involved in the bond, and then the name or abbreviation ofthe reducing saccharide (e.g., GlcNAc). The linkage between two sugarsmay be expressed, for example, as 2,3, 2→3, or (2,3). Each saccharide isa pyranose or furanose.

The term “recombinant” when used with reference to a cell indicates thatthe cell replicates a heterologous nucleic acid, or expresses a peptideor protein encoded by a heterologous nucleic acid. Recombinant cells cancontain genes that are not found within the native (non-recombinant)form of the cell. Recombinant cells can also contain genes found in thenative form of the cell wherein the genes are modified and re-introducedinto the cell by artificial means. The term also encompasses cells thatcontain a nucleic acid endogenous to the cell that has been modifiedwithout removing the nucleic acid from the cell; such modificationsinclude those obtained by gene replacement, site-specific mutation, andrelated techniques.

A “recombinant nucleic acid” refers to a nucleic acid that wasartificially constructed (e.g., formed by linking twonaturally-occurring or synthetic nucleic acid fragments). This term alsoapplies to nucleic acids that are produced by replication ortranscription of a nucleic acid that was artificially constructed. A“recombinant polypeptide” is expressed by transcription of a recombinantnucleic acid (i.e., a nucleic acid that is not native to the cell orthat has been modified from its naturally occurring form), followed bytranslation of the resulting transcript.

A “heterologous polynucleotide” or a “heterologous nucleic acid”, asused herein, is one that originates from a source foreign to theparticular host cell, or, if from the same source, is modified from itsoriginal form. Thus, a heterologous glycosyltransferase gene in aprokaryotic host cell includes a glycosyltransferase gene that isendogenous to (i.e., naturally present in) the particular host cell buthas been modified. Modification of the heterologous sequence may occur,e.g., by treating the DNA with a restriction enzyme to generate a DNAfragment that is capable of being operably linked to a promoter.Techniques such as site-directed mutagenesis are also useful formodifying a heterologous sequence.

The term “isolated” or “purified” is meant to refer to material which issubstantially or essentially free from components which otherwiseaccompany the material in its native state. For theoligosaccharide-containing binding moieties of the invention, forexample, a preparation of isolated or purified binding moieties includesa preparation that is substantially free of nucleic acids (e.g., genomicand other nucleic acids that are found in the microorganism cell thatsynthesized the oligosaccharide). Typically, isolated or purifiedoligosaccharides, glycoproteins, glycolipids, or other preparations froma recombinant microorganism of the invention are at least about 80%pure, usually at least about 90%, and preferably at least about 95% pureas measured by band intensity on a silver stained gel or other methodfor determining purity. Purity or homogeneity can be indicated by anumber of means well known in the art, such as polyacrylamide gelelectrophoresis of a protein or nucleic acid sample, followed byvisualization upon staining. For certain purposes high resolution willbe needed and HPLC or a similar means for purification utilized.

Detailed Description

This invention provides recombinant microorganisms that are useful forsynthesizing and delivering specific oligosaccharide structures to anorganism. The microorganisms synthesize oligosaccharides that, forexample, function as a binding moiety that, when administered to ananimal, competes with a ligand for binding to a receptor for the ligand.The oligosaccharides are synthesized in situ and displayed on thesurface of the microorganism. The oligosaccharide portion of the bindingmoiety includes one or more sugar residues that are attached to anacceptor moiety by glycosyltransferases. At least one of theglycosyltransferases that are involved in synthesis of theoligosaccharide is encoded by an exogenous nucleic acid which is presentin the microorganism.

Upon administration to a human or other animal, the recombinantmicroorganisms of the invention can function as a delivery vehicle forthe surface-displayed oligosaccharides, which are delivered to a targetsite, such as an endothelial or epithelial tissue, in the animal. Theoligosaccharides can then exert an effect, such as binding to a receptoron a cell, thus inhibiting binding of another ligand for the cell (e.g.,a bacteria or virus, a toxin, a cell involved in inflammation, etc.).The use of microorganisms to carry oligosaccharides provides severaladvantages over previously available methods for deliveringoligosaccharide-based molecules to humans and other animals. Forexample, digestive enzymes that are present in certain host environments(e.g., the small intestine) can cleave free oligosaccharides, reducingtheir utility for treatment of infections and other conditions in moredistal regions of the gut. The invention circumvents this problem byvirtue of having the oligosaccharides carried by microorganisms. Forgastrointestinal applications, for example, the microorganismspreferably are strains that are resistant to conditions found in thegut.

An illustrative example of the invention involves a recombinant deliverymicroorganism that displays a chimeric lipopolysaccharide structure, theterminal sugars of which constitute a Shiga toxin receptor mimic. Theserecombinant delivery microorganism are effective at protectingsusceptible cells from attack by the Shiga toxin whose receptor theymimic. The use of a chimeric carbohydrate moiety such aslipopolysaccharide means that the endogenous transport machinery of thedelivery microorganism is used to appropriately display the receptormimic. The effectiveness of providing a receptor mimic on the surface ofa recombinant microorganism has ramifications in relation to a broaderrange of toxins than simply Shiga toxins; it also has ramifications forother toxins such as those discussed above whose action requiresrecognition of oligosaccharide receptors. Additionally the receptors foradhesins of certain pathogenic microorganisms (including bacteria,viruses and parasites, for example) are structurally similar toreceptors for certain toxins, and the provision of receptors for suchadhesins on a microorganism surface should have a protective effect byinterfering with the pathogen and/or its capacity to infect host cells.Moreover the chimeric carbohydrate still has a protective effect ifsecreted or released into the environment, not being attached to thedelivery microorganism surface. This invention also opens the way to usenon-chimeric carbohydrates as competitive inhibitors for toxins andpathogenic micro-organisms.

Recombinant Microorganisms

The invention provides recombinant microorganisms that synthesizespecific oligosaccharide structures that have a desired biologicalactivity, e.g., binding to a receptor, toxin, or other molecule. Themicroorganisms can also be used to carry the oligosaccharide structuresto a target site (e.g., an animal membrane). There may be inherentadvantages in having the receptor formed by microorganism in situ,including cost, capacity of the delivery microorganism to undergomultiplication in the gut, expression of a high density of receptormimics on the cell surface, and display of the receptor mimic in anappropriate conformation.

Suitable microorganisms include, for example, bacteria, fungi, yeast,and other cells that include an acceptor moiety that can serve as asubstrate for a glycosyltransferase or other enzyme involved insynthesis of the desired oligosaccharide. The microorganisms preferablydisplay the oligosaccharide on a surface of the microorganism, or inanother form that is exposed to the surrounding environment afteradministration.

The microorganisms include at least one heterologous or exogenousnucleic acid that encodes one of the glycosyltransferases or otherenzymes that is involved in synthesizing the oligosaccharide structure.More than one exogenous nucleic acids can be present in a singlemicroorganism.

The glycosyltransferases or other enzymes add or remove a sugar from anacceptor molecule. The acceptor molecule can be a glycolipid,glycoprotein, or other carbohydrate that is anchored in the deliverymicroorganism, for example, capsular polysaccharides of eithergram-negative or gram-positive bacteria, or teichoic acids andlipoteichoic acids of gram-positive bacteria. In another form theacceptor molecule is one that is normally secreted into the externalmilieu, such as exopolysaccharides of either Gram-negative orGram-positive bacteria.

1. Glycosyltransferases

The recombinant cells of the invention contain at least one heterologousgene that encodes a glycosyltransferase or other enzyme that is involvedin oligosaccharide synthesis. Many glycosyltransferases are known, asare their polynucleotide sequences. See, e.g., “The WWW Guide To ClonedGlycosyltransferases,” (www.vei.co.uk/TGN/gt guide.htm).Glycosyltransferase amino acid sequences and nucleotide sequencesencoding glycosyltransferases from which the amino acid sequences can bededuced are also found in various publicly available databases,including GenBank, Swiss-Prot, EMBL, and others.

Glycosyltransferases that can be employed in the cells of the inventioninclude, but are not limited to, galactosyltransferases,fucosyltransferases, glucosyltransferases,N-acetylgalactosaminyltransferases, N-acetylglucosaminyltransferases,glucuronyltransferases, sialyltransferases, mannosyltransferases, andoligosaccharyltransferases. These glycosyltransferases include thoseobtained from both eukaryotes and prokaryotes. Many mammalianglycosyltransferases have been cloned and expressed and the recombinantproteins have been characterized in terms of donor and acceptorspecificity and they have also been investigated through site directedmutagenesis in attempts to define residues involved in either donor oracceptor specificity (Aoki et al. (1990) EMBO. J. 9: 3171-3178;Harduin-Lepers et al. (1995) Glycobiology 5(8): 741-758; Natsuka andLowe (1994) Current Opinion in Structural Biology 4: 683-691; Zu et al.(1995) Biochem. Biophys. Res. Comm. 206(1): 362-369; Seto et al. (1995)Eur. J. Biochem. 234: 323-328; Seto et al. (1997) J. Biol. Chem. 272:14133-141388).

In some embodiments, the glycosyltransferase is a fucosyltransferase. Anumber of fucosyltransferases are known to those of skill in the art.Briefly, fucosyltransferases include any of those enzymes which transferL-fucose from GDP-fucose to a hydroxy position of an acceptor sugar. Insome embodiments, for example, the acceptor sugar is a GlcNAc in aβGal(1→4)βGlcNAc group in an oligosaccharide glycoside. Suitablefucosyltransferases for this reaction include the knownGalβ(1→3,4)GlcNAc α(1→3,4)fucosyltransferase (FTIII E.C. No. 2.4.1.65)which is obtained from human milk (see, Palcic, et al., CarbohydrateRes. 190:1-11 (1989); Prieels, et al., J. Biol. Chem. 256:10456-10463(1981); and Nunez, et al., Can. J. Chem. 59:2086-2095 (1981)) and theGalβ(1→4)GlcNAc α(1→3)fucosyltransferases (FTIV, FTV, FTVI, and FTVII,E.C. No. 2.4.1.65) which are found in human serum. A recombinant form ofGalβ(1→3,4)GlcNAc α(1→3,4)fucosyltransferase is also available (see,Dumas, et al., Bioorg. Med. Letters 1:425-428 (1991) andKukowska-Latallo, et al., Genes and Development 4:1288-1303 (1990)).Other exemplary fucosyltransferases include α1,2 fucosyltransferase(E.C. No. 2.4.1.69). Enzymatic fucosylation may be carried out by themethods described in Mollicone, et al., Eur. J. Biochem. 191:169-176(1990) or U.S. Pat. No. 5,374,655.

In another group of embodiments, the glycosyltransferase is agalactosyltransferase. When a galactosyltransferase is used, thereaction medium will preferably contain, in addition to the cell thatcontains the exogenous galactosyltransferase gene, UDP-Gal and/or amechanism for its synthesis, an oligosaccharide acceptor moiety, and adivalent metal cation. Exemplary galactosyltransferases include α(1,3)galactosyltransferases (E.C. No. 2.4.1.151, see, e.g., Dabkowski et al.,Transplant Proc. 25:2921 (1993) and Yamamoto et al. Nature 345:229-233(1990), bovine (GenBank j04989, Joziasse et al. (1989) J. Biol. Chem.264:14290-14297), murine (GenBank m26925; Larsen et al. (1989) Proc.Nat'l. Acad. Sci. USA 86:8227-8231), porcine (GenBank L36152; Strahan etal (1995) Immunogenetics 41:101-105)). Another suitable α2,3galactosyltransferase is that which is involved in synthesis of theblood group B antigen (EC 2.4.1.37, Yamamoto et al. (1990) J. Biol.Chem. 265:1146-1151 (human)). Also suitable for use in the methods andrecombinant cells of the invention are α(1,4) galactosyltransferases,which include, for example, EC 2.4.1.90 (LacNAc synthetase) and EC2.4.1.22 (lactose synthetase) (bovine (D'Agostaro et al (1989) Eur. J.Biochem. 183:211-217), human (Masri et al. (1988) Biochem. Biophys. Res.Commun. 157:657-663), murine Nakazawa et al. (1988) J. Biochem.104:165-168), as well as E.C. 2.4.1.38 and the ceramidegalactosyltransferase (EC 2.4.1.45, Stahl et al. (1994) J. Neurosci.Res. 38:234-242). Other suitable galactosyltransferases include, forexample, al, α1,2 galactosyltransferases (from e.g., Schizosaccharomycespombe, Chapel et al (1994) Mol. Biol. Cell 5:519-528).

Sialyltransferases are another type of glycosyltransferase that isuseful in the recombinant cells of the invention. Examples ofsialyltransferases that are suitable for use in the present inventioninclude ST3Gal III (preferably a rat ST3Gal III), ST3Gal IV, ST3Gal I,ST6Gal I, ST3Gal V, ST6Gal II, ST6GalNAc I, ST6GalNAc II, and ST6GalNAcIII (the sialyltransferase nomenclature used herein is as described inTsuji et al. (1996) Glycobiology 6: v-xiv). An exemplaryα(2,3)sialyltransferase referred to as α(2,3)sialyltransferase (EC2.4.99.6) transfers sialic acid to the non-reducing terminal Gal of aGalβ1→3Glc disaccharide or glycoside. See, Van den Eijnden et al., J.Biol. Chem., 256:3159 (1981), Weinstein et al., J. Biol. Chem.,257:13845 (1982) and Wen et al., J. Biol. Chem., 267:21011 (1992).Another exemplary α2,3-sialyltransferase (EC 2.4.99.4) transfers sialicacid to the non-reducing terminal Gal of the disaccharide or glycoside.See, Rearick e al., J. Biol. Chem., 254:4444 (1979) and Gillespie etal., J. Biol. Chem., 267:21004 (1992). Further exemplary enzymes includeGal-β-1,4-GlcNAc α-2,6 sialyltransferase (See, Kurosawa et al. Eur. J.Biochem. 219: 375-381 (1994)).

Other glycosyltransferases that can be used the recombinant host cellsof the invention have been described in detail. In particular, theglycosyltransferase can also be, for instance, glucosyltransferases,e.g., Alg8 (Stagljov et al., Proc. Natl. Acad. Sci. USA 91:5977 (1994))or Alg5 (Heesen et al. Eur. J. Biochem. 224:71 (1994)),N-acetylgalactosaminyltransferases such as, for example, α(1,3)N-acetylgalactosaminyltransferase, β(1,4)N-acetylgalactosaminyltransferases (Nagata et al. J. Biol. Chem.267:12082-12089 (1992) and Smith et al J. Biol Chem. 269:15162 (1994))and polypeptide N-acetylgalactosaminyltransferase (Homa et al J. BiolChem. 268:12609 (1993)). Suitable N-acetylglucosaminyltransferasesinclude GnTI (2.4.1.101, Hull et al., BBRC 176:608 (1991)), GnTII, andGnTIII (Ihara et al. J. Biochem. 113:692 (1993)), GnTV (Shoreiban et al.J. Biol. Chem. 268: 15381 (1993)), O-linkedN-acetylglucosaminyltransferase (Bierhuizen et al. Proc. Natl. Acad.Sci. USA 89:9326 (1992)), N-acetylglucosamine-1-phosphate transferase(Rajput et al. Biochem J.285:985 (1992), and hyaluronan synthase.Suitable mannosyltransferases include α(1,2) mannosyltransferase, α(1,3)mannosyltransferase, β(1,4) mannosyltransferase, Dol-P-Man synthase,OCh1, and Pmt1.

Prokaryotic glycosyltransferases are also useful in the recombinantcells. Such glycosyltransferases include enzymes involved in synthesisof lipooligosaccharides (LOS), which are produced by many gram negativebacteria. The LOS typically have terminal glycan sequences that mimicglycoconjugates found on the surface of human epithelial cells or inhost secretions Preston et al. (1996) Critical Reviews in Microbiology23(3): 139-180). Thus, the use of such enzymes is particularly usefulfor making a recombinant delivery microorganism that mimics thesereceptor glycoconjugates and thus blocks binding of the pathogenicLOS-containing organisms. Such enzymes include, but are not limited to,the proteins of the rfa operons of species such as E. coli andSalmonella typhimurium, which include a β1,6 galactosyltransferase and aβ1,3 galactosyltransferase (see, e.g., EMBL Accession Nos. M80599 andM86935 (E. coli); EMBL Accession No. S56361 (S. typhimurium)), aglucosyltransferase (Swiss-Prot Accession No. P25740 (E. coli), anβ1,2-glucosyltransferase (rfaJ)(Swiss-Prot Accession No. P27129 (E.coli) and Swiss-Prot Accession No. P19817 (S. typhimurium)), and anβ1,2-N-acetylglucosaminyltransferase (rfaK)(EMBL Accession No. U00039(E. coli). Other glycosyltransferases for which amino acid sequences areknown include those that are encoded by operons such as rfaB, which havebeen characterized in organisms such as Klebsiella pneumoniae, E. coli,Salmonella typhimurium, Salmonella enterica, Yersinia enterocolitica,Mycobacterium leprosum, and the rhl operon of Pseudomonas aeruginosa.

Also suitable for use in the cells of the invention areglycosyltransferases that are involved in producing structurescontaining lacto-N-neotetraose,D-galactosyl-β-1,4-N-acetyl-D-glucosaminyl-β-1,3-D-galactosyl-β-1,4-D-glucose,and the P^(k) blood group trisaccharide sequence,D-galactosyl-α-1,4-D-galactosyl-β-1,4-D-glucose, which have beenidentified in the LOS of the mucosal pathogens Neisseria gonnorhoeae andN. meningitidis (Scholten et al. (1994) J. Med. Microbiol. 41: 236-243).The genes from N. meningitidis and N. gonorrhoeae that encode theglycosyltransferases involved in the biosynthesis of these structureshave been identified from N. meningitidis immunotypes L3 and L1(Jennings et al. (1995) Mol. Microbiol. 18: 729-740) and the N.gonorrhoeae mutant F62 (Gotshlich (1994) J. Exp. Med. 180: 2181-2190).In N. meningitidis, a locus consisting of three genes, lgtA, lgtB and lgE, encodes the glycosyltransferase enzymes required for addition of thelast three of the sugars in the lacto-N-neotetraose chain (Wakarchuk etal. (1996) J. Biol. Chem. 271: 19166-73). Recently the enzymaticactivity of the lgtB and lgtA gene product was demonstrated, providingthe first direct evidence for their proposed glycosyltransferasefunction (Wakarchuk et al. (1996) J. Biol. Chem. 271 (45): 28271-276).In N. gonorrhoeae, there are two additional genes, lgtD which addsβ-D-GalNAc to the 3 position of the terminal galactose of thelacto-N-neotetraose structure and lgtC which adds a terminal α-D-Gal tothe lactose element of a truncated LOS, thus creating the P^(k) bloodgroup antigen structure (Gotshlich (1994), supra.). In N. meningitidis,a separate immunotype L1 also expresses the P^(k) blood group antigenand has been shown to carry an lgtC gene (Jennings et al. (1995),supra.). Neisseria glycosyltransferases and associated genes are alsodescribed in U.S. Pat. No. 5,545,553 (Gotschlich). Anα1,3-fucosyltransferase gene from Helicobacter pylori has also beencharacterized (Martin et al. (1997) J. Biol. Chem. 272: 21349-21356).

In some embodiments, the recombinant delivery cells of the invention cancontain at least one heterologous gene that encodes a sulfotransferase.Such cells also produce the active sulfating agent3′-phosphoadenosine-5′-phosphosulfate (PAPS). Incorporation of one ormore sulfotransferase genes into a cell that also produces PAPS, eithernaturally or through the addition of the PAPS cycle regenerationenzymes, provides one with cells that can sulfate oligosaccharides orpolysaccharides. Suitable sulfotransferase include, for example,chondroitin-6-sulphotransferase (chicken cDNA described by Fukuta et al.(1995) J. Biol. Chem. 270:18575-18580; GenBank Accession No. D49915),glycosaminoglycan N-acetylglucosamine N-deacetylase/N-sulphotransferase1 (Dixon et al. (1995) Genomics 26:239-241; UL18918), andglycosaminoglycan N-acetylglucosamine N-deacetylase/N-sulphotransferase2 (murine cDNA described in Orellana et al. (1994) J. Biol. Chem.269:2270-2276 and Eriksson et al. (1994) J. Biol. Chem. 269:10438-10443,human cDNA described in GenBank Accession No. U2304).

Glycosyltransferase nucleic acids, and methods of obtaining such nucleicacids, are known to those of skill in the art. Glycosyltransferasenucleic acids (e.g, cDNA, genomic, or subsequences (probes)) can becloned, or amplified by in vitro methods such as the polymerase chainreaction (PCR), the ligase chain reaction (LCR), the transcription-basedamplification system (TAS), the self-substained sequence replicationsystem (SSR). A wide variety of cloning and in vitro amplificationmethodologies are well-known to persons of skill. Examples of thesetechniques and instructions sufficient to direct persons of skillthrough many cloning exercises are found in Berger and Kimmel, Guide toMolecular Cloning Techniques, Methods in Enzymology 152 Academic Press,Inc., San Diego, Calif. (Berger); Sambrook et al. (1989) MolecularCloning—A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring HarborLaboratory, Cold Spring Harbor Press, NY, (Sambrook et al.); CurrentProtocols in Molecular Biology, F. M. Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel); Cashion etal., U.S. Pat. No. 5,017,478; and Carr, European Patent No. 0,246,864.Examples of techniques sufficient to direct persons of skill through invitro amplification methods are found in Berger, Sambrook, and Ausubel,as well as Mullis et al. (1987) U.S. Pat. No. 4,683,202; PCR Protocols AGuide to Methods and Applications (Innis et al., eds) Academic PressInc. San Diego, Calif. (1990) (Innis); Arnhein & Levinson (Oct. 1, 1990)C&EN 36-47; The Journal Of NIH Research (1991) 3: 81-94; (Kwoh et al.(1989) Proc. Nat'l. Acad. Sci. USA 86: 1173; Guatelli et al. (1990)Proc. Nat'l. Acad. Sci. USA 87, 1874; Lomell et al. (1989) J. Clin.Chem., 35: 1826; Landegren et al., (1988) Science 241: 1077-1080; VanBrunt (1990) Biotechnology 8: 291-294; Wu and Wallace (1989) Gene 4:560; and Barringer et al. (1990) Gene 89: 117.

DNA that encodes glycosyltransferase proteins or subsequences, as wellas DNA that encodes the enzymes involved in formation of nucleotidesugars described below, can be prepared by any suitable method asdescribed above, including, for example, cloning and restriction ofappropriate sequences or direct chemical synthesis by methods such asthe phosphotriester method of Narang et al. (1979) Meth. Enzymol. 68:90-99; the phosphodiester method of Brown et al. (1979) Meth. Enzymol.68: 109-151; the diethylphosphoramidite method of Beaucage et al. (1981)Tetra. Lett., 22: 1859-1862; and the solid support method of U.S. Pat.No. 4,458,066. In one preferred embodiment, a nucleic acid encoding aglycosyltransferase can be isolated by routine cloning methods. Anucleotide sequence of a glycosyltransferase as provided in, forexample, GenBank or other sequence database can be used to provideprobes that specifically hybridize to a glycosyltransferase gene in agenomic DNA sample, or to a glycosyltransferase mRNA in a total RNAsample (e.g., in a Southern or Northern blot). Once the targetglycosyltransferase nucleic acid is identified, it can be isolatedaccording to standard methods known to those of skill in the art (see,e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2ndEd., Vols. 1-3, Cold Spring Harbor Laboratory; Berger and Kimmel (1 987)Methods in Enzymology, Vol. 152: Guide to Molecular Cloning Techniques,San Diego: Academic Press, Inc.; or Ausubel et al. (1987) CurrentProtocols in Molecular Biology, Greene Publishing andWiley-Interscience, New York).

A glycosyltransferase nucleic acid can also be cloned by detecting itsexpressed product by means of assays based on the physical, chemical, orimmunological properties. For example, one can identify a clonedglycosyltransferase nucleic acid by the ability of a polypeptide encodedby the nucleic acid to catalyze the transfer of a monosaccharide from adonor to an acceptor moiety. In a preferred method, capillaryelectrophoresis is employed to detect the reaction products. This highlysensitive assay involves using either monosaccharide or disaccharideaminophenyl derivatives which are labeled with fluorescein as describedin Wakarchuk et al. (1996) J. Biol. Chem. 271 (45): 28271-276. Forexample, to assay for a Neisseria lgtC enzyme, either FCHASE-AP-Lac orFCHASE-AP-Gal can be used, whereas for the Neisseria lgtB enzyme anappropriate reagent is FCHASE-AP-GlcNAc (Id.).

As an alternative to cloning a glycosyltransferase gene, aglycosyltransferase nucleic acid can be chemically synthesized from aknown sequence that encodes a glycosyltransferase. Chemical synthesisproduces a single stranded oligonucleotide. This can be converted intodouble stranded DNA by hybridization with a complementary sequence, orby polymerization with a DNA polymerase using the single strand as atemplate. One of skill would recognize that while chemical synthesis ofDNA is often limited to sequences of about 100 bases, longer sequencesmay be obtained by the ligation of shorter sequences.

Alternatively, subsequences can be cloned and the appropriatesubsequences cleaved using appropriate restriction enzymes. Thefragments may then be ligated to produce the desired DNA sequence.

The glycosyltransferase-encoding nucleic acids can be cloned using DNAamplification methods such as polymerase chain reaction (PCR). Thus, forexample, the nucleic acid sequence or subsequence is PCR amplified,using a sense primer containing one restriction site (e.g., NdeI) and anantisense primer containing another restriction site (e.g., HindIII).This will produce a nucleic acid encoding the desiredglycosyltransferase sequence or subsequence and having terminalrestriction sites. This nucleic acid can then be easily ligated into avector containing a nucleic acid encoding the second molecule and havingthe appropriate corresponding restriction sites. Suitable PCR primerscan be determined by one of skill in the art using the sequenceinformation provided in GenBank or other sources. Appropriaterestriction sites can also be added to the nucleic acid encoding theglycosyltransferase protein or protein subsequence by site-directedmutagenesis. The plasmid containing the glycosyltransferase-encodingnucleotide sequence or subsequence is cleaved with the appropriaterestriction endonuclease and then ligated into an appropriate vector foramplification and/or expression according to standard methods.

Other physical properties of a polypeptide expressed from a particularnucleic acid can be compared to properties of known glycosyltransferasesto provide another method of identifying glycosyltransferase-encodingnucleic acids. Alternatively, a putative glycosyltransferase gene can bemutated, and its role as a glycosyltransferase established by detectinga variation in the structure of an oligosaccharide normally produced bythe glycosyltransferase.

In addition to, or instead of, glycosyltransferases, one can use adifferent enzyme that adds sugar residues to, or removes from, anacceptor molecule. Accordingly, the invention provides recombinantmicroorganisms that have an exogenous nucleic acid that encodes anenzyme such as a sialidase (e.g., trans-sialidase), mannosidase,galactosidase, glucosidase. Through use of such enzymes, an acceptormolecule can be modified to obtain an oligosaccharide structure thatexhibits the desired biological property.

In some embodiments, it may be desirable to modify theglycosyltransferase or accessory enzyme nucleic acids. One of skill willrecognize many ways of generating alterations in a given nucleic acidconstruct. Such well-known methods include site-directed mutagenesis,PCR amplification using degenerate oligonucleotides, exposure of cellscontaining the nucleic acid to mutagenic agents or radiation, chemicalsynthesis of a desired oligonucleotide (e.g., in conjunction withligation and/or cloning to generate large nucleic acids) and otherwell-known techniques. See, e.g., Giliman and Smith (1979) Gene 8:81-97,Roberts et al. (1987) Nature 328: 731-734. For example, one can modifythe glycosyltransferase gene to change the specificity or, for example,to stabilise a gene that might be subject to phase variation duringnormal cellular processes such as replication.

In a preferred embodiment, the recombinant nucleic acids present in thecells of the invention are modified to include preferred codons whichenhance translation of the nucleic acid in a selected organism (e.g.,yeast preferred codons are substituted into a coding nucleic acid forexpression in yeast).

2. Accessory enzymes involved in nucleotide sugar formation

In selecting the delivery microorganism it may be found that a suitablecomplement of endogenous glycosyltransferases are already present butthere is a surfeit of enzymes for the production of precursor nucleotidesugars, such enzyme could include epimerases, dehydrogenases,transmutases. The addition of one of these enzyme can be sufficient toprovide for a chimeric carbohydrate that is capable of acting as areceptor mimic, more commonly it is anticipated that a gene encoding anexogenous enzyme required for nucleotide precursor production may needto be introduced in addition to the one or more exogenous glycosyltransferases.

Accordingly, the recombinant microorganisms of the invention can alsoinclude, in addition to or in place of the nucleic acid encoding aglycosyltransferase or other enzyme involved in oligosaccharidesynthesis, at least one heterologous nucleic acid that encodes anaccessory enzyme. Accessory enzymes include, for example, those enzymesthat are involved in the formation of a nucleotide sugar. The accessoryenzyme can be involved in attaching the sugar to a nucleotide, or can beinvolved in making the sugar or the nucleotide, for example. Examples ofnucleotide sugars that are used as sugar donors by glycosyltransferasesinclude, for example, GDP-Man, UDP-Glc, UDP-Gal, UDP-GlcNAc, UDP-GalNAc,CMP-sialic acid, UDP-xylose, GDP-Fuc, GDP-GlcNAc, among others.

Accessory enzymes that are involved in synthesis of nucleotide sugarsare well known to those of skill in the art. For a review of bacterialpolysaccharide synthesis and gene nomenclature, see, e.g., Reeves etal., Trends Microbiol. 4: 495-503 (1996). The methods described abovefor obtaining glycosyltransferase-encoding nucleic acids are alsoapplicable to obtaining nucleic acids that encode enzymes involved inthe formation of nucleotide sugars. For example, one can use one ofnucleic acids known in the art directly or as a probe to isolate acorresponding nucleic acid from other organisms of interest.

3. Expression cassettes and host cells for expressing the fusionpolypeptides

A wide variety of microorganisms are useful for synthesis and/orcarrying of oligosaccharides according to the methods of the invention.To obtain expression of the glycosyltransferase and/or other enzymesthat are involved in synthesis of the binding moieties, the nucleicacids that encode the enzymes are placed under the control of a promoterthat is functional in the desired host cell. An extremely wide varietyof promoters are well known, and can be used in the expression vectorsof the invention, depending on the particular application. Ordinarily,the promoter selected depends upon the cell in which the promoter is tobe active. Other expression control sequences such as ribosome bindingsites, transcription termination sites and the like are also optionallyincluded. Constructs that include one or more of these control sequencesare termed “expression cassettes.”

Expression control sequences that are suitable for use in a particularhost cell are often obtained by cloning a gene that is expressed in thatcell. Commonly used prokaryotic control sequences, which are definedherein to include promoters for transcription initiation, optionallywith an operator, along with ribosome binding site sequences, includesuch commonly used promoters as the beta-lactamase (penicillinase) andlactose (lac) promoter systems (Change et al., Nature (1 977) 198:1056), the tryptophan (trp) promoter system (Goeddel et al., NucleicAcids Res. (1980) 8: 4057), the tac promoter (DeBoer, et al., Proc.Natl. Acad. Sci. U.S.A. (1 983) 80:21-25); and the lambda-derived P_(L)promoter and N-gene ribosome binding site (Shimatake et al., Nature(1981) 292: 128). The particular promoter system is not critical to theinvention, any available promoter that functions in prokaryotes can beused.

For expression of fusion polypeptides in prokaryotic cells other than E.coli, a promoter that functions in the particular prokaryotic species isrequired. Such promoters can be obtained from genes that have beencloned from the species, or heterologous promoters can be used. Forexample, a hybrid trp-lac promoter that functions in Bacillus inaddition to E. coil is described in WO9820111.

A ribosome binding site (RBS) is conveniently included in the expressioncassettes of the invention. An RBS in E. coli, for example, consists ofa nucleotide sequence 3-9 nucleotides in length located 3-11 nucleotidesupstream of the initiation codon (Shine and Dalgarno, Nature (1975)254:34; Steitz, In Biological regulation and development: Gene expression(ed. R. F. Goldberger), vol. 1, p. 349, 1979, Plenum Publishing, NY).

For expression of the fusion polypeptides in yeast, convenient promotersinclude GAL1-10 (Johnson and Davies (1984) Mol. Cell. Biol. 4:1440-1448)ADH2 (Russell et al. (1983) J. Biol. Chem. 258:2674-2682), PHO5 (EMBO J.(1982) 6:675-680), and MFa (Herskowitz and Oshima (1982) in TheMolecular Biology of the Yeast Saccharomyces (eds. Strathern, Jones, andBroach) Cold Spring Harbor Lab., Cold Spring Harbor, N.Y., pp. 181-209).Another suitable promoter for use in yeast is the ADH2/GAPDH hybridpromoter as described in Cousens et al., Gene 61:265-275 (1987). Forfilamentous fungi such as, for example, strains of the fungi Aspergillus(McKnight et al., U.S. Pat. No. 4,935,349), examples of useful promotersinclude those derived from Aspergillus nidulans glycolytic genes, suchas the ADH3 promoter (McKnight et al., EMBO J. 4: 2093 2099 (1985)) andthe tpiA promoter. An example of a suitable terminator is the ADH3terminator (McKnight et al.).

Either constitutive or regulated promoters can be used in themicroorganisms of the invention. Regulated promoters can be advantageousbecause the host cells can be grown to high densities before expressionof the glycosyltransferases is induced. High level expression ofheterologous proteins slows cell growth in some situations. An induciblepromoter is a promoter that directs expression of a gene where the levelof expression is alterable by environmental or developmental factorssuch as, for example, temperature, pH anaerobic or aerobic conditions,light, transcription factors and chemicals. Such promoters are referredto herein as “inducible” promoters, which allow one to control thetiming of expression of the glycosyltransferase or other enzyme involvedin nucleotide sugar synthesis. Such promoters can also be useful forsuppressing synthesis of the oligosaccharide until after the organismshave been administered. At a desired time a stimulus can be applied, sothat the oligosaccharide structure is synthesized after the deliverymicroorganism has arrived at a desired target site.

For E. coli and other bacterial host cells, inducible promoters areknown to those of skill in the art. These include, for example, the lacpromoter, the bacteriophage lambda P_(L) promoter, the hybrid trp-lacpromoter (Amann et al. (1983) Gene 25: 167; de Boer et al. (1983) Proc.Nat'l. Acad. Sci. USA 80: 21), and the bacteriophage T7 promoter(Studier et al. (1986) J. Mol. Biol.; Tabor et al. (1985) Proc. Nat'l.Acad. Sci. USA 82: 1074-1078). These promoters and their use arediscussed in Sambrook et al., supra. A particularly preferred induciblepromoter for expression in prokaryotes is a dual promoter that includesa lac promoter component linked to a promoter component obtained from agene or genes that encode enzymes involved in galactose metabolism(e.g., a promoter from a UDPgalactose 4-epimerase gene (galE)). The duallac-gal promoter, which is described in PCT Patent Application Publ. No.WO98/20111, provides a level of expression that is greater than thatprovided by either promoter alone.

DNA encoding the exogenous genes can be carried on a non-integratedvector such as a plasmid, selected to be stable within the deliverymicroorganism. Such vectors are known to those skilled in the art. Onebenefit in having the DNA in a non-integrated form is that a high copynumber of the encoding DNA can mean that where competitive addition ofsugars to an intermediate acceptor molecule is required, the enzymeencoded by the high copy number gene can prevail. An alternative is tohave the exogenous gene or genes incorporated into the microorganismchromosome. This tends to provide a greater measure of stability.

Methods for cloning a glycosyltransferase gene from one species toanother to make chimeric lipopolysacchacrides are described in, forexample, Phillips et al, (2000) J. Biol. Chem. 275:4747-4758 and AbuKwaik et al., (1991) Molec. Microbiol. 5:2475-2480.

Methods of Administering Recombinant Microorganisms

The invention also provides methods of administering the recombinantmicroorganisms, or oligosaccharide-containing fractions thereof, to ahuman or other animal. The microorganisms or fractions thereof can thusfunction as carriers for the oligosaccharides. The oligosaccharides arepreferably displayed on the surface of the microorganism, or areotherwise exposed to the environment after administration to the animal.Such exposure can result from the natural expression, or from processingof the microorganism after synthesis of the oligosaccharide. Forexample, a membrane preparation, lipopolysaccharide, glycoprotein,glycolipid, or other moiety that upon which an oligosaccharide can beattached can be administered. The preparation can be used unpurified, orpartially or completely purified. It is often desirable that thepreparation is at least substantially free of nucleic acids from themicroorganism.

The oligosaccharide-containing binding moieties can be delivered to, forexample, a mucosal surface of a mammal. Such mucosal surfaces include,for example, the gastrointestinal tract, the pulmonary and bronchialtissues, vaginal surfaces. The binding moieties can also be administeredto, for example, the eye, skin, etc.

The quantity administered is chosen to be sufficient is have the desiredeffect. For example, in some applications the amount of binding moietyadministered is sufficient to reduce adherence of a pathogen or a toxinproduced by a pathogen to a mucosal or other surface.

The delivery microorganism is chosen to be non-harmful whenadministered. It is also possible that a pathogen or an organism withpotentially adverse health effects is used, and this would beadministered in an attenuated form, or may be administered in a killedform. The killing of the microorganism is to be conducted under standardconditions that maintain the chimeric carbohydrate molecule, and inparticular the receptor mimic intact. Examples of methods of providingkilled microorganism include, but are not limited to, treatment withchemical agents such as formalin, thiomersal, or streptomycin or otherbactericidal antibiotic, or exposure to heat or UV iradiation. Bacterialghosts generated by induction of a bacteriophage lysis protein may alsobe a suitable delivery vehicle for receptor mimics, as would liposomesincorporating the chimeric LPS. In the alternative purified membranevesicles (MVs) can be used. MVs are naturally released by gram negativebacteria and can be described as blebs of the outer membrane. Purifiedlipopolysaccharide can be used however preferably the deliverymicroorganism expresses mutations which decrease endotoxin activity ofLPS, and such mutations can include phoP^(c)(67) or msbB (waaN) (68,69).

The delivery microorganism can be utilised in a live form so that it canmultiply in vivo at least to a limited extent, thereby producing morereceptor mimics, so that smaller doses can be effective. However,particularly for administration to humans, it is preferable that an atleast partially purified preparation of the oligosaccharides is used.

The stomach of an individual presents a considerable barrier to theintroduction of microorganism and acid labile macromolecules. It can bedesirable to provide for some acid resistance. For example a particlecan be delivered that has a protective coating, such as those that arecommonly used in the delivery of pharmaceuticals, which can then beselectively released either in the small intestine, large intestine orboth. Such coatings and capsules are well known. An alternative approachcan be to make the delivery microorganism more acid tolerant, forexample the delivery microorganism could have a constitutiveacid-tolerance response. It is sometimes desired to grow the deliverymicroorganism in media that enhance their capacity to pass the acidbarrier of the stomach; for example the culture can be grown under lowpH conditions to induce acid tolerance.

Additionally one can provide for some resistance to anti microbialactivity that is presented by the resident microflora. Thus for examplethe delivery microorganism could be modified or chosen to be resistantto the major families of colicins (for example Col E1, E2 and E3) by theintroduction of, for example, the btuB mutation.

The choice of delivery microorganism is wide insofar as what is requiredis that the organism is capable of expressing the chimeric carbohydratemolecule being delivered to the gut. The delivery mode can be in aprotected environment such as by being coated or inside a capsule, andthus the organism need not necessarily be acid resistant. It is alsopossible that the delivery microorganism need not survive in the gut andthese measures may also be useful for delivery of kllled microorganismor partially or fully purified carbohydrates carrying the receptormimic. All that is required is that the receptor mimic be exposed insufficient quantities to adsorb toxin or the pathogenic organism. It ispreferred however that the organism does survive and grow and thereforepresents an increasing level of the chimeric carbohydrate foradsorption, accordingly it is desired that the organism is resistant toconditions found in the gut and thus is an enteric organism.

The delivery microorganism can be selected from the genuses Escherichiaand Salmonella, and more preferably Escherichia coli and Salmonellaenterica sv typhimurium. However, certain other bacterial genera may beconvenient to use, notably those such as Acidophilus, Lactobacillus,Lactococcus or Bifidobacterium, which being food bacteria, are known tobe safe to administer orally to humans, and are also capable of survivalin the gut. These later organisms have, in common with Escherichia andSalmonella, been relatively well characterised.

To be more effective it is desired that the delivery microorganismproperly exposes the receptor mimic in as effective a manner aspossible. Accordingly it is desired that the delivery microorganism doesnot produce or has a limited capacity to produce any of the following:O-antigen, a slime layer, capsule or exopolysaccharide, where suchcarbohydrates are not the carrier of the receptor mimic. These moleculesmight otherwise mask the receptor mimic.

It is also desirable that the microorganism expressing the receptormimic colonizes the gastrointestinal tract for only a limited period oftime, so as not to perturb normal receptor-ligand interactions, ordisturb the state of immune tolerance which prevents elicitation ofpotentially deleterious anti-receptor antibodies. Incorporation of aninducible suicide gene may help eliminate the recombinant microorganismfrom the host when its presence is no longer necessary. An example ofsuch as system is the stochastic lethal containment system described byKlemm et al. (42).

The recombinant microorganisms of the invention find use human (inpharmaceutical use, and also for veterinary use. For example,microorganisms that display an appropriate oligosaccharide for animalpathogens are useful to treat livestock and other animals of interest.Such animals include, but are not limited to, humans, pigs, cows,horses, canines, felines, chickens, turkeys, goats, rabbits, sheep,geese, ducks.

In a second form of the second aspect the invention provides arecombinant microorganism expressing one or more exogenous sugartransferases and an acceptor molecule, said one or more exogenous sugartransferases being specific for the transfer of one or more sugarresidues represented progressively from a non reducing terminal end of areceptor of either a toxin or an adhesin of a pathogenic organism, saiddelivery microorganism expressing the exogenous sugar transferases, andprogressively transferring said one or more sugar resides onto theacceptor molecule to thereby form a chimeric carbohydrate molecule withan exposed receptor mimic, said exposed receptor mimic capable ofbinding the toxin or the adhesin.

Suitable excipients for the delivery of the receptor mimic are readilyascertainable, and examples can be found in Remington's PharmaceuticalSciences, Mack Publishing Company, 16th Edition, 1980. Similarly methodsfor delivery by packing in capsules or within a matrix such as a tabletcan be found in the same reference.

Uses of the Recombinant Microorganisms

The recombinant microorganisms of the invention are useful for a widevariety of applications upon administration to a human or other mammal.

1. Toxins and Adhesins

The invention provides methods of reducing the amount of a toxin or apathogenic organism from an environment. These methods involveintroducing at least an inoculum of a recombinant microorganism into theenvironment. The recombinant microorganism carries a binding moiety thatmimics a receptor of either the toxin or an adhesin of the pathogenicorganism is exposed to the environment for contact with the toxin oradhesin. The oligosaccharide-containing binding moiety can be, forexample, anchored to the outer surface of the microorganism, or secretedinto the environment.

For example, a recombinant microorganism that displays a chimericcarbohydrate molecule that functions as a mimic of a receptor for atoxin, or for an adhesin of a pathogenic organism, can be administeredto block binding of the toxin and/or pathogenic organism to thereceptor. The chimeric receptor can be chosen to bind toxins, adhesinsof enteric bacterial or viral or parasitic pathogens, or lectinsassociated with these organisms.

The toxins include those made in the gut and can be selected from, butnot limited to, a group comprising shiga toxins, clostridial toxins,cholera toxins, E. coli enterotoxins, and Staphylococcal enterotoxins.

As an illustrative example, the shiga toxins can be selected from thegroup comprising, Stx, Stx1, Stx2, Stx2c, Stx2d, and Stx2e. In the casewhere the shiga toxin is Stx, Stx1, Stx2, Stx2c, or Stx2d, the receptormimic preferably is formed by a terminal sugar moiety ofGalα[1→4]Galβ[1→4]Glc. The one or more transferases is either an α1→4galactosyl transferase capable of forming an α1→4 bond with a galactoseresidue bonded by a β1→4 bond to a glucose on the acceptor molecule, orboth an α1→4 galactosyl transferase and α1→4 galactosyl transferasecapable of forming a β1→4 bond to a glucose on the acceptor molecule.While it might be desirable that the exogenous glycosyl transferases addsugars to a sugar residue of the acceptor molecule, it may be adequatethat the glycosyl transferases compete with an endogenous transferase,and that the acceptor molecule is a partially completed endogenousoligosaccharide or polysaccharide.

Where the shiga toxin is Stx2e the an alternative oligosaccharide moietyof the receptor for a terminal chain is suggested to beGalNAcβ[1→3]Galα[1→4]Galβ[1→4]Glc. The one or more transferases areselected to transfer one or more sugars selected from the terminalportion of the receptor for Stx2e. In addition to those transferaseslisted above, the one or more transferases are selected to include aβ1→3GalNAc transferase capable of forming a β1→3 bond with a galactoseresidue.

For Stx, Stx1, Stx2, Stx2c, and Stx2d receptors, the N-terminal sugartransferase gene can be selected from the group comprising lgtC of N.meningitidis or N. gonorrhoeae and the penultimate terminal sugartransferase gene may be selected from the group comprising lgtE of Nmeningitidis or N. gonorrhoeae; Haemophilus influenzae strain Rd is alsoknown to contain genes encoding enzymes with analogous functions.

In the case of K88ad adhesin its receptor includes the lactoneotetraoseseries of glycolipids. These carbohydrates are produced by N.Meningitidis, N. gonorrhoeae, and some strains of Haemophilus influenzaeas one of their variable LOS components, and genetic material from thesestrains can be used to construct a recombinant receptor mimic.

The clostridial toxins include, for example, tetanus toxin, botulinumtoxin, C. difficile toxins A and B. The natural receptor for C.difficile toxin A is known to contain a Galβ[1→4]GlcNAc moiety. Also,however, Galα[1→3]Galβ[1→4]GlcNAc- or Galα[1→3]Galβ[1→4]Glc- are alsoknown to interact with toxin A. The receptor for botulinum toxin is alsobelieved to be a sialic acid containing glycoprotein or glycolipidpresent on neurons.

The cholera toxins include cholera toxin and E. coli heat labileenterotoxin types I and II. The receptor for cholera toxin and E. coliheat labile enterotoxin type I is the ganglioside G_(M1), the structurebeing as follows:

The receptor mimic can be chosen from 2 or more adjacent sugar residuesin the configuration as set out immediately above.

The receptor mimic can be selected from any one of the receptors set outin Table 1.

TABLE 1 Glycosyl structures of receptors for toxins and adhesinsGalα[1→4]Galβ[1→4]Glc Shiga toxin Stx1, Stx2, Stx2c, Stx2d, Stx2e,uropathogenic E. coli pap pili Galα[1→4]Galβ uropathogenic E. coli pappili GalNAcβ[1→3]Galα[1→4]Galβ[1→4]Glc Shiga toxin Stx2e Galβ[1→4]GlcNAcC. difficile toxin A Galα[1→3]Galβ[1→4]Glc C. difficile toxin AGalα[1→3]Galβ[1→4]GlcNAc C. difficile toxin AGalβ[1→4]GlcNAcβ[1→3]Galβ[1→4]Glc C. difficile toxin A Glcα[1→6]Glc— C.difficile toxin B Glcα[1→6]Glcα[1→6]Glc— C. difficile toxin B NeuNAc— C.botulinum toxin

Vibrio cholera toxin (CT), E. coli heat labile enterotoxin Type 1Galβ[1→3]GalNAcβ[1→4]Galβ[1→4]Glc— Enterotoxigenic E. coli CFA adhesin,porcine enterotoxigenic E. coli K88ad fimbriae adhesin GalNAcβ[1→4]GalEnterotoxigenic E. coli CS3 pili adhesin GalNAc— Entamoeba histolyticumtrophozoite adherence Gal— E. histolyticum adherence NeuGc→GM₃ Porcinerotavirus adherence NeuNAc→GM₃ Porcine rotavirus adherence

The receptor mimic can be a mimic of the natural receptor for adhesinsor toxins produced by micro-organism selected from a group of generacomprising the following:—Escherichia, Salmonella, Shigella,Citrobacter, Helicobacter, Yersinia, Vibrio, Aeromonas, Campylobacter,Pseudomonas, Pasteurella, Neisseria, Haemophilus; Moraxella, Klebsiella,Staphylococcus, Streptococcus, Clostridium, Bacteriodes as well asviruses including rotavirus.

Additional examples of oligosaccharides that can be displayed on themicroorganism, and examples of their use, are listed in Table 2.

TABLE 2 Terminal Example Sugar Oligosaccharide Inhibits adhesion of:Reference Sialic acid Ganglioside Staphylococcus pneumonia, H. vanAlphen et al. (1991) influenzae, H. parainfluenza, Infect. Immun. 59:4473 Pseudomonas Sialic acid LSTd, LSTc Staphylococcus pneumonia, H.Simon et al., WO influenzae, H. parainfluenza, 96/40169 PseudomonasSimon et al., U.S. Pat. No. 5,736,533 Idanbaan-Heikkila et al. (1997) J.Infect. Dis. 176: 704-712 Galactose LNnT Staphylococcus pneumonia, H.Simon et al., WO influenzae, H. parainfluenza, 96/40169 PseudomonasSimon et al., U.S. Pat. No. 5,736,533 Idanbaan-Heikkila et al. (1997) J.Infect. Dis. 176: 704-712 Galactose asialo-N-linked Chlamydiatrachomatis Kuo et al. (1996) J. oligosaccharides Clin. Invest. 98:2813-2818 Mannose Oligomannose Acanthamoeba (e.g., adhesion Cao et al.(1998) J. to cells in eye) Biol. Chem. 273: 15838 Fucose 2′FucosidesCandida ablicans Cameron, B J (1996) linked to Gal Infect. Immun. 64:891 Fucose 2′ and 3′ Helicobacter pylori (e.g., Falk et al. (1993) Proc.fucosides (e.g., adhesion to gastric cells) Nat'l. Acad. Sci. USALewis^(y), Lewis^(b) 90: 2035 GalNAc Many pulmonary pathogenic bacteriaKrivan et al. (1988) (see list in reference) Proc. Nat'l. Acad. Sci. USA85: 6157 Fucose or Blood groups A, Candida ablicans Critchley et al.(1987) J. GlcNAc B, H Gen. Microbiol. 133: 637; Matei et al. (1997) Rev.Roum. Biochim. 34: 123 GalNAc Many Streptococcus pneumoniae Tuomanen etal. (WO (e.g., adhesion to pulmonary 95/33467) cells) Sialic acidEnterotoxigenic E. coli Sjoberg et al. (1988) Biochem. J. 255: 105Schwertmann et al. (1999) J. Pediatric Gastroenterology and Nutrition28: 257 Sialic acid Mycoplasma pneumonia (e.g., Roberts et al. (1989) J.ear infections, pneumonia) Biol. Chem. 264: 9289 Krivan et al., U.S.Pat. No. 5,089,479

E. coli heat-labile enterotoxin Barra et al. (1992) Mol. Cell. Biochem.115: 63-70

E. coli heat-labile enterotoxin Barra et al. (1992) Mol. Cell. Biochem.115: 63-70 Sialic acid 3′ and 6′ Adhesion to stomach Zopf et al.,sialylosides (e.g., U.S. Pat. No. sialyllactose) 5,514,660 Sialic acidSLe^(x) and SLe^(a) Inhibition of selectin-mediated Paulson et al.,inflammation U.S. Pat. No. 5,753,631 Ratcliffe et al., U.S. Pat. No.5,576,305 DeFrees et al., U.S. Pat. No. 5,811,404 Sialic acidStreptococcus sanguis (e.g., Nesser et al. (1995) adhesion to humanbuccal Glycobiology 5: 97 epithelial cells) Gal LNnT, LNT, etc.Streptococcus pyrogenes (e.g., Gaffar et al., adhesion to pharyngealcells) U.S. Pat. No. 5,401,723 Sialic acid (incl. N-Ac- Influenza (e.g.,adhesion to Eisen et al. (1997) sialic acid; N-Gc- cells) Virology 232:19 sialic acid, and 9- Gambaryan et al. (1997) Oac-sialic acid Virology232: 345 Connor et al. (1994) Virology 205: 17 Herrler et al. (1987)Virology 159: 102 Fucose 2′-fucosides such E. coli and Vibrio choleraePrieto et al., WO as 2′- (e.g., adhesion to 99/56754 fucosyllactosegastrointestinal cells) Fucose 2′-fucosides Campylobacter jejuni (e.g.,Newburg, D S (1997) adhesion to gastrointestinal Am. Symp. Nutritionalcells) Sci. 980S-984S Stable enterotoxin (ST) of E. coli (binding oftoxin to cells) Sialic acid Paramyxovirus Klenk et al. (1997) Nova ActaLeopoldina NF75, NR. 301, 131 9-OAc-sialic Human coronavirus Klenk etal. (1997) acid Bovine coronavirus Nova Acta Leopoldina NF75, NR. 301,131 GalNAc GalNAc(β1,3) Parvovirus Brown et al. (1993) Gal(α1,4)GalScience 262: 114-117 Sialic acid 3′ or 6′- Cells and molecules involvedin U.S. patent application Ser. No. sialosides inflammation, irritation(e.g., of 09/123,251 (pending) skin, intestine, etc)

In one form the acceptor molecule is a molecule which comprises at leastsome sugar units, these sugar units are normally exposed to the outsideof the delivery microorganism. A preferred acceptor molecule is alipopolysaccharide (LPS) molecule, and in which case the acceptormolecule is all or a portion of the core of the lipopolysaccharide.

In the case where the two terminal sugars of the chimeric receptor bindsto Stx, Stx1, Stx2, Stx2c, and Stx2d, it may be preferred that theacceptor molecule has a terminal glucose. The acceptor molecules can bethe LPS core of waaO mutants of Escherichia coil R1 wherein a terminalglucose is presented in the completed acceptor molecule of the mutantconcerned. Alternatively a terminal glucose need not be provided, andthe choice of bacterium based on LPS core structure is wide indeed.Specific examples of core structure that are suitable as acceptormolecules include but are not limited to the following, Escherichia coliR1, including waaO mutants thereof, Escherichia coli K-12 and variouswaa mutants thereof Salmonella typhimurium LT2, and various waa mutantsthereof. It will be understood however that the range of molecules fromwhich the acceptor molecule can be chosen is wide.

The elucidation of the nature of the oligosaccharide receptors is anongoing endeavour, and more receptors for toxins and adhesins are beingdefined as time goes on. Selecting appropriate carbohydrate structurescan be achieved by searching through a database of known carbohydratestructures, such as Carbbank, which is available over the internet;CDrom versions are also available from NBRF, National BiomedicalResearch Foundation, 3900 Reservoir Road, NW, Washington D.C. 20007 USA.Selecting appropriate nucleic acid sequences for expression of thedesired glycosyl transferases can be achieved by searching through adatabase of genes encoding glycosyl transferases available over theinternet, such as CAZy. This database is administered by AFMB-CNRS acontact being at 31 Chemin Joseph Aiguier F-13402 Marseille Cedex 20(France). An alternative is to search for structures in the ChemicalAbstracts. The search concerned will identify the sugar specificity ofthe transferase, the sugar to which it binds, the nature of the bond,and the overall nature of the acceptor molecule. Thus where the acceptormolecule is a LPS then a transferase specific for LPS will be preferred.The gene encoding the transferase of interest can be either madesynthetically or alternatively the gene may be isolated from anappropriate organism either by direct cloning methods or by PCRamplification methods and incorporated into an expression vector. Adatabase suitable for searching of enzymes that may be used to providefor nucleotide precursors such as epimerases, dehydrogenases,transmutases and the like can be found at the following internetaddress: wit.mcs.anl.gov/WIT2/.

Toxins are often produced by pathogens in precursor form, and in thatcase it may be the precursor form that can recognise the toxin receptorreferred to in this specification.

2. Other Applications

The recombinant microorganism of the invention, and oligosaccharidesobtained from the microorganism, are also useful as carriers foroligosaccharides that have a cosmeceutical, diagnostic orpharmacological application. The recombinant microorganisms can, forexample, be used to reduce the number of pathogenic organisms fromenvironments such as the oral cavity, the urogenital tract or othermucosal surface, or external environments such as waterways.

Examples of such oligosaccharides and uses that involve modulation ofcell adhesion and/or interactions are listed in Table 3.

TABLE 3 Possible Compound Mechanism Use Reference Sialyl lactose nervegrowth Japan patent factor-like agent appl. No. for treating aging07258093A of nervous system Sialic acid Hair growth and Japan patentderivatives (e.g., protection; appl. no. sialosides from suppression ofJP0727929 cows milk) dandruff and itchiness Sialyl lactose Inhibitsimmune Rheumatoid U.S. Pat. No. complex forma- arthritis 5,614,374 tion(in vitro) Sialyl lactose EGF receptor Treatment for WO 97/03701(multivalent) antagonist cancer Sialyl lactose Reduces Psoriasis andinflammation acute dermatitis Sialyl lactose Maintain Japan patentganglioside appl. content in brain JP09315981 Sialyl lactose ReducesReduction in skin U.S. Pat. Appl. inflammation reddening (e.g., No.(selectin and/or cosmetics, sun 09/123,251 sialoadhesin screens, babycare (pending) inhibitor) products, OTC anti-inflammatory agents, acneagents Sialyl lactose Sialoadhesins Immuno- Crocker et al. (inhibitionof suppression (1997) adhesion) (macrophages, Glycoconjugategranulocytes) J. 14: 601 sialoadhesin, May et al. MAG, CD33, (1997) SMP(Swann cell Protein Sci. myelin protein) 6: 717

In other applications, the recombinant microorganism of the invention,and oligosaccharides obtained from the microorganism, are useful forgastrointestinal administration Examples of such uses are listed inTable 4.

TABLE 4 Possible Compound Mechanism Use Reference Sialyl lactosePromotes mineral absorption Sialyl lactose Bifidobactor Maintain gutIdota et al., growth promoter normal flora Biosci. Biotech. Biochem. 58:1720 Sialyl lactose Cholera toxin Prevents binding Idota et al.antagonist of toxin, reducing (1995) Biosci. diarrhea Biotech. Biochem.59: 417 Sialyl lactose Rotavis adhesion Prevents viral See, e.g., Taiyoinhibitors binding, thereby Kaguki (1995) reducing diarrhea J. Agric.Food Chem. 43: 858 Sialyl lactose Heat-labile E. coli Prevents toxinU.S. Pat. No. toxin adhesion adhesion, limiting 5,627,163 inhibiteddiarrhea Sialyl lactose Prevents toxin Bacterial toxin U.S. Pat. No.(and sialic acid adhesion neutralizer, limit- 5,260,380 derivatives) ingor preventing diahhrea N-Gc-sialyl K99 toxin Reduces calf Lanne et al.lactose adhesion scours (1995) Biochemistry 34: 1845 Willemsen andDeGraaf (1993) Infect Immun. 61: 4518 Sialyl lactose Prevents ReducesSjoberg et al. enterotoxigenic incidence (1988) E. coli cell of diarrheaBiochem adhesion through J. 255: 105 the CS2 lectin Sialyl lactoseIntestinal Japan patent metabolism appl. no. improving agent JP10029945

In other applications, the recombinant microorganism of the invention,and oligosaccharides obtained from the microorganism, are useful fornasopharyngeal administration. Examples of such uses are listed in Table5.

TABLE 5 Possible Compound Mechanism Use Reference Sialyl lactoseInfluenza A and Reduces influenza Frank et al. (a) 2,3 and 2,6 Badhesion symptoms by (1997) J. Mol. (b) muitivalent preventing viralModel. 3: 408 adhesion Gambaryan et al. (1997) Virology 232: 345Synthesome, WO 98/14215 Eisen et al. (1997) Virology 237: 19 Sialyllactose Paramyxovirus Treats pig blue Reyes-Leyva et eye disease al.(1993) Arch. Virol. 133: 195 Sialyl lactose C. albicans Reduces Annaixet al. adhesion incidence of (1990) FEMS lung infection Microbiol.Immun. 64: 147 Sialyl lactose Polyoma virus Thilo and adhesion Harrison(1996) Structure 4: 183 Bauer et al. (1995) J. Virol. 69: 7925 Sialyllactose Streptococcus U.S. Pat. No. adhesion 5,401,723 e.g., pyogenesReduces Nesser et al. and mutans incidence of (1995) cavitiesGlycobiology e.g., sanguis Reduces 5: 97 incidence of WO 95/33467gingivitis Sialyl LNnT S. pneumonia Reduces otitis (sialyl lactose)adhesion media and respiratory tract infections Sialyl lactose B.pertussis Reduces toxin adhesion whooping cough coughing symptoms Sialyllactose Expectorant U.S. Pat. No. 4,698,332 Sialyl lactose and H.influenza otitis media and Lock van derivatives (e.g., adhesionmeningitis Alphen et al. SLNnT) (1991) Infect. Immun 59: 4473 WO96/40169 Sialyl lactose Prevents Reduces infection Bouchara et al. (andsialic acid) Aspergillus and severity of (1997) Infect. fumigatusbinding infections in and Immun. to lamina and diseases such as 65: 2717fibrinogen assinusitis and broncho- putmonary aspergillosis Sialyllactose Mycoplasma Treat or prevent Krivan, U.S. (multivalent)pneumoniae/ laryngitis and Pat. No. hominus pneumonia, 5,089,479adhesion to cervical Roberts et al. laminin and genital (1989) Biol.diseases Chem. 65: 2717 Tokojenko et al. (1994) Microbiol Zh. 56: 3Sialyl LNnT Pneumococcal Pneumonia Idanpaan- pneumonia Heikkila et al.adhesion (1997) J. Infect. Dis. 176: 704 WO 95/33467 Sialyl lactoseGargle to prevent Japan Patent colds appl. no. JP90268129

The recombinant microorganisms of the invention are also useful to carrybacteriocins. Bacteriocins are described in, for example, WO9506736.

The invention also encompasses a method of testing for the presence of atoxin or a pathogenic microorganism, including the steps of contacting asample with a recombinant microorganism, or a purified oligosaccharidefrom a microorganism. Either the carbohydrate or the sample can beimmobilised, if desired. Upon binding of the toxin or pathogenicmicroorganism to the oligosaccharide, the unbound toxin or pathogenicmicroorganism can be removed by washing. A detection agent is then usedto detect bound toxin or pathogenic microorganism.

EXAMPLES

The following examples are offered to illustrate, but not limit theinvention.

Example 1

Background

Shiga toxin- (Stx-) producing strains of Escherichia coli (STEC) areimportant causes of diarrhoea and haemorrhagic colitis (HC) in humans.This can lead to potentially fatal systemic sequelae, such as haemolyticuraemic syndrome (HUS) which is the leading cause of acute renal failurein children (1, 2, 3, 4). Certain other Enterobacteriaceae are alsoknown to produce Stx and cause serious gastrointestinal disease inhumans. The most notable of these is Shigella dysenteriae type 1, thecausative agent of bacillary dysentery, which is frequently associatedwith Stx-induced systemic sequelae, including HUS (1). Indeed, it is theprincipal cause of HUS in parts of Africa and Asia (4). Stx-producingCitrobacter freundii has also been shown to cause diarrhea and HUS inhumans, including one outbreak in a German child-care centre (4).

The mortality rate for HUS is 5-10%; other acute complications includestroke, diabetes mellitus, and necrotising colitis necessitatingcolectomy. The Center for Disease Control and Prevention (Atlanta, Ga.)has estimated that the annual cost for acute care of patients with STECdisease in the USA is in the range of $1-2 billion, with approximately500 deaths each year (3). In addition, up to a third of survivorssustain permanent renal impairment and may eventually requiretransplantation (2). Estimates of the on-going cost of management ofthese long-term complications are not available.

STEC are commonly found in the intestines of livestock, and humaninfections usually result from consumption of contaminated meat or dairyproducts; fruit and salad vegetables contaminated with manure, andcontaminated drinking or swimming water are also common STEC vehicles.In addition, approximately 20% of all cases of STEC disease are believedto result from person to person transmission (3, 6). STEC belonging toover 100 O:H serotypes have been associated with human disease. However,those belonging to serogroup O157 (particularly O157:H7) are the mostprevalent causes of HUS and account for the majority of the majorfood-bourne outbreaks in the United States, Europe and Britain (2, 3).It is likely, however, that the epidemiological data on the overallincidence of STEC disease and serotype prevalence has been skewed byunderdetection of cases caused by non-O157 STEC, which are much moredifficult to detect (3, 4). In recent years there have been a number oflarge outbreaks of STEC disease in North America, the UK, Europe, Japanand Australia Such outbreaks have the potential to overwhelm acute carefacilities, even in developed countries with sophisticated health-caresystems. The largest (Sakai, Japan, May-June 1996) involved over 8,000cases of HC (600 requiring hospitalization) and 107 cases of HUS.Another outbreak involving over 500 people and 20 deaths occurred inScotland in December 1996.

Stx is a compound toxin, consisting of an enzymatically active A subunit(a RNA-N-glycosidase), which inhibits eukaryotic protein synthesis, anda pentameric B subunit responsible for binding to glycolipid receptorsin target cell membranes (2). Two major classes of Stx (Stx1 and Stx2)have been distinguished, both by serological methods, as well as by DNAsequence analysis. Individual STEC strains may produce toxins belongingto either or both of the two major Stx classes. However, substantialvariation in amino acid sequence occurs within both the Stx1 and Stx2groups (7, 8, 9, 10,11,12). Within the Stx2 class, several subtypes havebeen distinguished on the basis of differences in biological properties.All Stx types associated with human disease recognize the sameglycolipid receptor, globotriaosyl ceramide (Gb₃), which has thestructure Galα[1→4]Galβ[1→4]Glc-ceramide (13). Vero (African greenmonkey kidney) cells express large amounts of Gb₃ on their surface,consequently, this cell line is highly susceptible to Stx, and Verocytotoxicity is the generally recognised standard assay for Stxactivity. However, one particular subgroup of Stx2 variants, designatedStx2c, share specific B subunit amino acid differences with respect toclassical Stx2 (Asp₁₆-Asn and Asp₂₄-Ala), which correlate with asomewhat reduced binding affinity for the receptor Gb₃ and reduced invitro cytotoxicity for HeLa cells (14). A separate subgroup, designatedStx2d, is distinguished from other Stx toxins by increased cytotoxicityfor Vero cells after incubation in the presence of either mouse proximalsmall intestinal mucus or human colonic mucus. Activation appears to bea function of the A subunit, because the B subunit is identical toStx2c, which was not activatable. Activatable Stx2d toxins examined todate share two A subunit amino acid differences with respect to Stx2c(Ser₂₉₁ and Glu₂₉₇), although these alone may not necessarily besufficient for activation, as they are also found in some Stx2 subtypeswhich are not activatable (15). A final major Stx2 subtype is Stx2e,which is produced by STEC associated with piglet oedema disease. This isa serious, frequently fatal illness affecting piglets at the time ofweaning, and is characterized by neurological symptoms including ataxia,convulsions and paralysis; oedema is typically present in the eyelids,brain, stomach, intestine and mesentery of the colon (16). It isassociated with particular STEC serotypes (most commonly O138:K81,O139:K82 and O141:K85, which are not associated with human disease.These particular STEC strains also cause post-weaning diarrhoea inpiglets. Stx2e has a different glycolipid receptor specificity fromother members of the Stx family, recognising globotetraosyl ceramide(Gb₄; GalNAcβ[1→3]Galα[1→4]Galβ[1→4]Glc-ceramide) preferentially overGb₃ (17). Two amino acid differences in the Stx2e B subunit (Gln₆₄ andLys₆₆) are critical for this altered specificity, which impacts on thetissue tropism of the toxin, thereby accounting for the distinctiveclinical presentation of oedema disease (18).

The pathological features seen in severe human STEC disease (HC and HUS)are directly attributable to the Stx toxins, which are essential forvirulence. Pathogenesis of disease initially involves colonization ofthe gut by the STEC; the bacteria do not invade the gut epithelium, butlocally produced Stx is absorbed into the circulation, and the toxinthen targets specific tissues in accordance with their Gb₃ content. Inhumans Gb₃ is found in highest concentrations in renal tissue, and inmicrovascular endothelial cells (particularly in the kidneys, gut,pancreas and brain), thereby accounting for the distinct clinical andpathological features of HUS (microangiopathic haemolytic anaemia,thrombocytopoenia and renal failure).

There is increasing evidence that STEC strains vary in their capacity tocause serious disease in humans, and that this, at least in part, is afunction of the type and/or amount of Stx produced. Indeed, up to1000-fold differences in the cytotoxicities of various STEC isolatesfrom humans have been reported. Moreover, patients infected with STECproducing Stx2 are more likely to develop serious complications such asHUS than those infected with STEC producing Stx1 (19,20). The linkbetween Stx2 production and HUS may be a direct consequence of increasedin vivo toxicity of Stx2. Indeed, human renal microvascular endothelialcells have been shown to be far more susceptible to the cytotoxic actionof Stx2 than Stx1 (21). This is consistent with studies employing astreptomycin-treated mouse model of toxin-induced renal tubular damage.Oral challenge with E. coli K-12 carrying cloned Str2 genes, but notSix1 genes was capable of inducing fatal tubular damage (22).

The availability of rapid and sensitive methods for diagnosis of STECinfection early in the course of disease has created a window ofopportunity for therapeutic intervention. Indeed, during two outbreakswhich have occurred in Adelaide we diagnosed STEC infection in patientsby PCR almost a week before symptoms of HUS became apparent. Theincreased awareness that occurs during major outbreaks is likely toresult in more patients presenting during the early (diarrhoeal) stage,and when a source of infection has been identified and publicised,persons exposed to the contaminated product may come forward beforesymptoms appear. An opportunity also exists to treat close contacts ofpersons with proven or suspected STEC infection (e.g. family members,children in child-care centres, school classmates, etc.), to preventthem from developing serious disease. Antibiotic therapy iscontraindicated for STEC infection because of the risk of increasingfree Stx in the gut lumen through release of cell-associated toxin andinduction of toxin gene expression. There are also concerns thatantibiotic therapy might disturb gut flora and result in overgrowth bythe STEC. Administration of antimotility agents is also contraindicated(2,4).

During the early stages of human infections, STEC may colonize the gutat high levels (>90% of aerobic flora), exposing the host to sustainedhigh concentrations of Stx and increasing the likelihood of systemiccomplications. However, as disease progresses, the numbers of STECdecrease markedly, and may even be undetectable in patients who havealready progressed to HUS. Western blot analysis using convalescent serafrom HUS patients suggests that the elimination of STEC from the gutduring the latter stages of HUS is probably a consequence of localimmune responses to STEC surface antigens (23). Clearly, in cases ofnatural STEC infection, the immune response occurs too slowly to preventStx-induced complications. Thus, in vivo binding or neutralization ofStx is a potentially important therapeutic strategy. Substances capableof binding Stx in the gut can also play a role as an adjunct toantibiotic therapy.

AU Stx types affecting humans recognise the same glycolipid receptor(Gb₃), and at least one strategy exploiting this interaction has beendeveloped. This agent, called Synsorb-Pk, consists of chemicallysynthesised Galα[1→4]Galβ[1→4]Glc- (the trisaccharide component of Gb₃)covalently linked via an 8 carbon spacer to silica particles derivedfrom diatomaceous earth. Synsorb-Pk is capable of binding andneutralizing Stx1 and Stx2 in STEC culture extracts, and in faeces frompatients with HC and HUS, although binding of Stx2-related toxins isless efficient than for Stx1 (24,25). 1 mg of Synsorb-Pk has been shownto be capable of binding 93% of a 0.5 ng aliquot of radioiodinated Stx1(24). Other in vitro studies using purified toxins indicate that thesaturation binding capacity of 1 mg of Synsorb-Pk is approximately 5 ngof purified Stx1 or Stx2 (26). In this latter study coincubationexperiments indicated that 1 mg of Synsorb-Pk could protect 50% of cellsin tissue culture from 2 ng of Stx1 or 0.4 ng of Stx2. A phase Iclinical trial did not detect any adverse effects associated with oraladministration, and Synsorb-Pk retained its Stx-binding capacity afterpassage through the human gastrointestinal tract (27). Results of arandomized, double-blind trial vs placebo in children with STECdiarrhoea indicated that oral administration of approximately 500 mgSynsorb-Pk per kg per day reduced the relative risk of progression toHUS by approximately 40%, but only if administered within 3 days ofonset of disease (25).

Materials and Methods, Results and Discussion

In this example, the invention resides in construction of a harmlessrecombinant microorganism capable of incorporating the trisaccharideGalα[1→4]Galβ[1→4] Glc- into the outer core region of itslipopolysaccharide, such that a mimic of the natural host receptor forthe toxin is displayed on the bacterial surface. This recombinantmicroorganism, either live or killed, is capable of binding all Stxtypes associated with human disease. This microorganism binds Stx2,Stx2c, and Stx2d toxins as effectively as it does Stx1. This is a veryimportant property, since the Stx2-related toxins have greater toxicityfor human renal microvascular endothelial cells than Stx1, and strainsproducing Stx2 are more frequently associated with HUS than thoseproducing only Stx1, as described above. Moreover, this finding wasunexpected, given that the synthetic product Synsorb-Pk has a lowerbinding affinity for Stx2-related toxins than it has for Stx1 (seeabove). In addition, the recombinant microorganism is capable of bindingthe oedema disease-associated toxin Stx2e.

Procedure for Construction of Recombinant Bacteria Expressing theTrisaccharide Galα[1→4]Galβ[1→4 Glc- on their Surface

In order to locate a source of bacterial genes encoding biosynthesis ofthe trisaccharide component of the receptor for Stx, we conducted asearch of carbohydrate structure databases. This revealed thatGalα[1→4]Galβ[1→4]Glc- is found in the outer core region of thelipooligosaccharides (LOS) of Neisseria meningitidis (28) and N.gonorrhoeae (29), as well as in the LOS of certain strains ofHaemophilus influenzae (30). LOS are the major glycolipids expressed onthe surface of these bacteria, and are analogous to the LPS of otherGram-negative bacterial genera such as Escherichia and Salmonella. LOSand LPS have many structural features in common. Lipid A is the commonhydrophobic moiety, and this forms the outer leaflet of the outermembrane. This is linked to the inner core oligosaccharide, whichconsists largely of heptose and KDO (3-deoxy-D-manno-oct-2-ulosonicacid). The general structural features of lipid A and the inner coreoligosaccharide are highly conserved amongst diverse bacterial species,as they are important for outer membrane stability (31,32). The outerregion of the core oligosaccharide comprises hexoses, which are linkedto the inner core by a variety of highly specific glycosyl transferases.Thus, this region varies in structure from species to species and evenwithin a given bacterial strain (see below). In Enterobacteriaceae, anantigenic repeat-structure O-polysaccharide is attached to the distalend of the outer core oligosaccharide to form high molecular weight LPS.However, in genera such as Neisseria, this does not occur, resulting ina much lower molecular weight LOS (31,32). Pathogenic species ofNeisseria have also been shown to sialylate their LOS, an importantvirulence trait, as this confers a high degree of resistance to thebactericidal activity of serum (29,31).

Another important property of N. gonorrhoeae and N. meningitidis is thecapacity to undergo phase variation which alters the structure (andhence the antigenicity) of the outer core oligosaccharide, therebyavoiding host immune responses. In both species, one of the alternativeLOS immunotypes has an outer core consisting ofGalα[1→4]Galβ[1→4]Glc-→(referred to as immunotype L1). Phase variationoccurs spontaneously and at such high frequency that in any givenculture, cells expressing LOS with different outer core oligosaccharideimmunotypes will be present. This, as well as the fact that they arepathogens, precludes use of these Neisseria sp. as in vivo toxin-bindingagents. Genetic characterization of the region of the N. gonorrhoeaechromosome encoding outer core biosynthesis has provided a molecularexplanation for the LOS phase variation phenomenon (33,34). This regioncontains five glycosyl transferase genes, lgtA, lgtB, lgtC, lgtD andlgtE, arranged as an operon. Three of these genes (lgtA, lgtC and lgtD)contain poly-G tracts within the respective open reading frame, whichrenders them highly susceptible to slipped strand mispairing duringreplication. Such slippage results in frame shift mutations andconcomitant premature termination of translation of the respectivespecific glycosyl transferase (34). Thus, the actual outer coreoligosaccharide structure, and hence immunotype, will depend on which ofthe lgt genes are encoding functional enzymes at a given point in time.The exact specificity of the five gonococcal glycosyl transferases hasbeen determined by mutational analysis (34). From this we have deducedthat expression of the L1 immunotype LOS requires functional lgtE andlgtC genes, which encode the transferases responsible for linking theα-galactosyl and β-galactosyl residues onto the glucose residue attachedto the distal end of the inner core oligosaccharide. For this to happenin Neisseria, lgtA must be non-functional, as this gene encodes a highlyactive N-acetylglucosamine transferase which adds GlcNAc to the outercore in lieu of the terminal α-Gal. The products of lgtB and lgtD, onthe other hand, do not appear to interfere with synthesis of immunotypeL1, presumably because these transferases have absolute specificity foracceptor oligosaccharides containing GlcNAc (34).

Sequence data for the entire Igt region from both N. gonorrhoeae and N.meningitidis are available on the GenBank database (accession numbersU14554 and U65788, respectively). This was used to design theoligonucleotide primers shown in Table 6 to direct amplification of lgtCand IgtE genes:

TABLE 6 Primers used for amplification of IgtC and IgtE genesRestriction site Primer Sequence (5′-3′) inserted (underlined) LGTCFGAACAGGAATTCGGCAAGATTATTGTGCC EcoRI (SEQ ID. NO:1) LGTCRTACGTCGGATCCCGTCTGAAGGCTTCAGAC BamHI (SEQ ID. NO:2) LGTEFGCCCTTGGATCCACCGCAGCTATTGAAACC BamHI (SEQ ID. NO:3) LGTERCCATTTAAGCTTTTAATCCCCTATATTTTACAC HindIII (SEQ ID. NO:4)

The lgtC and lgtE genes were PCR amplified using primer pairsLGTCF/LGTCR and LGTEF/LGTER, respectively, with N. Meningitidis and N.gonorrhoeae DNA as template, respectively (the lgtC and lgtE genes fromthe two species are approximately 95% identical). These PCR productswere cloned into the vector pK184 (35) after digestion of both vectorand PCR product with EcoRl/BamHI or BamHI/HindIII, respectively, andtransformed into E. coli K-12. Since the lgtC gene is one of those witha poly-G tract, it was necessary to mutate this region to stabilizeexpression of the encoded transferase. The DNA sequence of N.meningitidis lgtC from nt 157-171 of the open reading frame isCGGGGGGGGGGGGGT (SEQ ID NO:5), which encodes the amino acid sequenceArg-Gly-Gly-Gly-Gly (SEQ ID NO:6). This region of the lgtC gene clonedin pK184 was mutated to CGTGGCGGTGGCGGT (SEQ ID NO:7) by overlapextension PCR. This involved separate PCR amplification of overlapping5′ and 3′ portions of the cloned lgtC gene. The 5′ portion was amplifiedusing the universal M13 reverse sequencing primer and another with thesequence ATATTACCGCCACCGCCACGCAAATTGGCGGC (SEQ ID NO:8), whereas the 3′portion was amplified using the universal M13 forward sequencing primerand another with the sequence AATTTGCGTGGCGGTGGCGGTAATATCCGCTT (SEQ IDNO:9). The two PCR products were then purified, aliquots were mixed, andfull length lgtC with the desired modifications was amplified by PCRusing the M13 forward and reverse primers. The PCR product was digestedwith EcoRI/BamHI and once again cloned into similarly digested pK184,and subjected to sequence analysis to confirm mutagenesis of the poly Gtract. This eliminated the possibility of slipped strand mispairingwithout affecting the amino acid sequence of the encoded protein. Themutated lgtC gene was then excised from the pK184 construct withEcoRI/BamHI and cloned into the compatible restriction sites in thepK184 derivative containing lgtE. This places the lgtC and lgtE genes intandem in pK184, in the same orientation as the vector lac promoter. Thealtered lgtC gene sequence take from nt 157-171 of the open readingframe is as follows, the altered nucleotides are shown in bold andunderlined TTTGCGTGGCGGTGGCGGTAATAT (SEQ ID NO:10).

The recombinant pK184:lgtCE plasmid was then transformed into a suitableE. coli host. In the first instance we used a derivative of E. coil R1(designated CWG308) which has a non-polar insertion mutation in the waaOgene, resulting in expression of an LPS core consisting of just theinner core plus Glc linked to the terminal heptose residue (36). Thisstructure is very similar to the natural substrate for the galactosyltransferase LgtE, and so CWG308 is an appropriate host for expression oflgtCE. A derivative of E. coli K-12 with mutations in waaO and waaB isalso a suitable host for expression of lgtCE, as it has the samelipopolysaccharide core structure as CWG308. This host has an additionaladvantage in that it has been proven to be safe for oral administrationto humans in very high doses (37). Extensive studies carried out in theearly 1980s demonstrated that although it is capable of growth in thehuman gut, it can not establish long-term, high level colonization, asit lacks adhesins found in pathogenic strains of E. coli (37).

Transformation of CWG308 with pK184/lgtCE resulted in synthesis of LPSwith an outer core oligosaccharide containing a terminalGalα]1→4]Galβ[1→4]Glc→epitope, as judged by reactivity on dot-immunoblotwith a monoclonal antibody specific for the N. meningitidis L1immunotype. Moreover, CWG30b:pK184/lgtCE is capable of directly bindingand neutralizing Stx, as detailed below.

Procedure for Testing the Capacity of Recombinant Bacteria to NeutraliseStx

CWG308 and CWG308:pK184/lgtCE were grown overnight at 37° C. in LB broth(supplemented with IPTG and also with 25 μg/ml kanamycin in the case ofCWG308:pK184/lgtCE), harvested by centrifugation, washed and resuspendedin phosphate-buffered saline (PBS) at a density of approximately 1×10⁹CFU/ml (equivalent to approximately 2 mg dry weight of cells per ml). Inthe first instance, French pressure cell (FPC) lysates of freshovernight LB broth cultures of the following E. Coli strains were usedas a source of Stx.

TABLE 7 E. coli strains used as a source of stx Strain Description Ref.EDL933 Wild type O157:H7 STEC producing (38) Stx1 and Stx2 JM109:pJCP521E. coli JM109 with Stx_(2c) cloned in pBluescript (11) JM109:pJCP525 E.coli JM109 with Stx₁ cloned in pBluescript  (9) JM109:pJCP539 E. coliJM109 with Stx₂ cloned in pBluescript (39) JM109:pJCP542 E. coli JM109with Stx_(2d) cloned in pBluescript  (8) 128/12 Wild type piglet oedemadisease STEC producing Stx2e

FPC lysates of each of the above cultures were filter-sterilized and 0.5ml aliquots were incubated with 1 ml of CWG308 or CWG308:pK184/lgtCEsuspension, or PBS, for 1 hour at 37° C. with gentle agitation. Themixtures were then centrifuged and filter-sterilized. Twelve serial2-fold dilutions were prepared in tissue culture medium (Dulbecco'sModified Eagles Medium buffered with 20 mM HEPES, and supplemented with2 mM L-glutamine, 50 IU/ml penicillin and 50 μg/ml streptomycin),commencing at a dilution of 1:20. Fifty μl of each dilution wastransferred onto washed Vero cell monolayers in 96well tissue culturetrays, and after 30 min incubation at 37° C., a further 150 μl ofculture medium was added to each well. Cells were examinedmicroscopically after 72 hours incubation at 37° C., and scored forcytotoxicity. The endpoint Stx titre was defined as the reciprocal ofthe highest dilution which still resulted in detectable cytotoxicity.Results for the various Stx extracts are shown below.

TABLE 8 Neutralization of Stx toxins % Stx neutralized by: CWG308: Stxsource Stx type Stx titre CWG308 pK184/lgtCE EDL933 Stx1 & Stx2   409600   99.6 JM109:pJCP521 Stx2c   10240 0   99.8 JM109:pJCP525 Stx1 >409600   99.6 JM109:pJCP539 Stx2   10240 0   98.4 JM109:pJCP542 Stx2d    12800 >99.2 128/12 Stx2e    1280 0   87.5

The capacity of killed CWG308:pK184/lgtCE cells to bind and neutralizeStx was also examined. Cell suspensions were killed by heating at 65° C.for 3 hours, or by treatment with 1% formaldehyde for 16 hours at 4° C.Capacity to neutralize cytotoxicity in FPC extracts containing Stx1 orStK2c was then compared with that for live CWG308:pK184/lgtCE cells,under the standard conditions described above. The Stx titres for theStx1 and Stx2c extracts used in this experiment were 40960 in bothcases. Heat-killed CWG308:pK184/lgtCE neutralized 93.7% of the Stx1 and96.8% of the Stx2c. Formaldehyde-killed CWG308:pK184/lgtCE neutralized99.6% of the Stx1 and 99.2% of the Stx2c. Live CWG308:pK184/lgtCE cellsneutralized 99.2% of the Stx1 and 99.6% of the Stx2c. Thus,heat-treatment slightly reduces the capacity of CWG308:pK184/lgtCE cellsto bind and neutralize Stx, but formaldehyde-killed CWG308:pK184/lgtCEcells are as effective as live cells. Whilst it is preferable to uselive CWG308:pK184/lgtCE from an efficiency point of view because of itscapacity to multiply in the gut, thereby increasing the number of cellscapable of binding Stx, formaldehyde-killed cells could be used incircumstances where administration of live cells is contraindicated(e.g. immunocompromised patients), or where regulatory approvalrequirements dictate otherwise.

Presence of both lgtC and lgtE genes in the recombinant plasmid wasessential, as CWG308 carrying derivatives of pK184 containing eitherlgtC or lgtE alone did not bind Stx toxin.

Strains with mutations in genes encoding outer core glycosyltransferases such that a rough LPS comprising the inner core plus Glclinked to the terminal heptose residue is produced can be expected to bepreferred hosts for expression of lgtCE. However, it is possible that ifexpression of these genes in the heterologous host is sufficient, thetwo encoded galactosyl transferases may compete with endogenoustransferases in host strains lacking outer core mutations. This maydirect biosynthesis of a modified LPS containing a Stx-binding epitope.This was examined by transforming a range of wild type and mutant E.coli and Salmonella typhimurium (S enterica sv typhimurium) LT2 strainswith pK184/lgtCE and examining the capacity to bind Stx. Experimentalconditions were the same as those used for the CWG308 derivative. FPClysates of E. coli JM109:pJCP525 and JM109:pJCP521 were used as a sourceof STx1 and Stx2c, respectively.

TABLE 9 Neutralization using live receptor mimics % Stx1 neutralized %Stx2c neutralized −pK184/ +pK184/ −pK184/ +pK184/ Strain IgtCE IgtCEIgtCE IgtCE E. coli K-12 C600 0 99.6 0 99.2 E. coli K-12 D21 0 99.2 098.4 E. coli K-12 D21e7 0 99.6 0 93.7 E. coli K-12 D21f1 0 98.4 0 98.4E. coli B BL21 0 0  0 0  S. typhimurium LT2 0 99.6 0 98.4 SL3748 S.typhimurium LT2 0 99.2 0 96.8 SL3750 S. typhimurium LT2 0 93.7 0 0 SL3769

All E. coli strains are reference to in (64), all Salmonella strains areobtainable from Salmonella Genetic Stock Centre accessible on theinternet at the following-address.www.acs.ucalgary.ca/˜kesander/index.html

Thus, host strains suitable for expression of lgtCE are not limited tostrains with mutations in outer core LPS synthesis. Vectors other thanpK184 may also be suitable for expression of these genes, includingthose with higher or lower copy number, different strength promoters(either constitutive or inducible), and those which utilize alternativeselection markers, e.g. alternative antibiotic resistance genes, ormarkers capable of complementing auxotrophic mutations, such as thyA⁺.Alternatively, the lgtC and lgtE genes could be integrated into the hostchromosome by allellic exchange using an appropriate suicide vector suchas pCACTUS, or others known to those skilled in the art.

Produce for Measuring Total Stx Binding Capacity of CWG308 pK184/lgtCF

To determine the total binding capacity of CWG308:pK184/lgtCE cells,suspensions containing 5×10⁸ CFU (1 mg dry weight) in PBS were incubatedat 37° C. for 1 hour with aliquots (ranging from 1 ng to 640 μg) ofpurified Stx1 and Stx2 (obtained from Toxin Technologies Inc., Florida,USA) in a final volume of 0.5 ml, and cytotoxicity was compared withthat for similar aliquots of toxin incubated with CWG308.

TABLE 10 Stx binding capacity of recombinant strains Amount of Stx %Stx1 neutralized % Stx2 neutralized 1 ng 99.98  99.95 5 ng 99.98  99.9520 ng 99.98  99.95 50 ng 99.95 99.9 100 ng 99.95 99.9 200 ng 99.95 99.9500 ng 99.2  99.9 1 μg 99.2  99.2 2 μg 99.2  99.2 4 μg 98.4  98.4 8 μg98.4  96.8 16 μg 98.4  96.8 32 μg 98.4  96.8 40 μg 98.4  96.8 80 μg98.4  93.7 160 μg 87.5  87.5 320 μg 50   50   640 μg 0   0 

From Table 10 it can be seen that the saturation Stx binding capacity of1 mg of CWG308:pK184/lgtCE cells is approximately 100 μg for both Stx1and Stx2. This binding capacity is more than 10,000 times greater thanthat claimed for Synsorb-Pk (25,26).

Procedure for Testing Capacity of Live CWG308:pK184/lgtCE Cells toProtect Mice from Fatal Infection with STEC

A streptomycin-treated mouse model of lethal Stx2-induced renal damagehas been described previously (12,22,40). Two wild type STEC strainswere used; B2F1 (which produces Stx2d), and 97MW1 (which produces Stx2).B2F1 is known to have very high virulence in this model; mice fed as fewas 10 organisms succumb (41). Two groups of 8 streptomycin-treatedBalb/C mice were challenged with approximately 1×10⁸ CFU of STEC B2F1;another two groups of 8 mice were challenged with STEC 97MW1. Mice werethen given oral doses of approximately 4×10⁹ CFU of either CWG308 orCWG308:pK184/lgtCE suspended in 60 μl of 20% sucrose, 10% NaHCO₃, twiceper day. The numbers of STEC, as well as-either CWG308 orCWG309:pK184/lgtCE, as appropriate, were monitored in faecal samplesfrom each group. One day (24 hours) after challenge, faecal pelletscontained approximately 10⁹ CFU of the respective STEC per g. Faecalpellets from groups which received CWG308:pK184/lgtCE also containedapproximately 10³ CFU of this strain. For both the B2F1 and 97W1 groups,all of the mice which received oral CWG308 died (median survival time 4days). However, all of the mice which received CWG308:pK184/lgtCEsurvived and were alive and well two weeks after challenge. Thisdifference in survival rate (8/8 vs 0/8) is highly significant(P<<0.005, Fisher exact test) and demonstrates unequivocally that oraladministration of CWG308:pK184/lgtCE is capable of preventing the fatalsystemic complications of STEC disease.

This example has now been published by the inventors in the followingjournal Nature Medicine 6; 265-270 (March 2000).

Example 2

Here we examine the capacity of oral administration of killedrecombinant cells to protect mice from otherwise fatal challenge with ahighly virulent STEC strain. We have also examined the effect ofdelaying commencement of therapy on protective efficacy.

Material and Methods

Bacterial strains and plamids.

E. coli CWG308 has been described previously (36) and was provided byChris Whitfield (Department of Microbiology, University of Guelph,Canada). Construction of plasmid pK184/lgtCE (referred to in thisexample as pJCP-Gb3) is as described in example 1. The Stx2-producingO113:H21 STEC strains 97MW1 and 98NK2 are both clinical isolates fromthe Women's and Children's Hospital, North Adelaide, South Australiawhich have been described previously (39). Spontaneousstreptomycin-resistant derivatives of these strains used in challengeexperiments were isolated by in vitro exposure to the drug. All E. colistrains were routinely grown in Luria-Bertani (LB) medium (44) with orwithout 1.5% Bacto-Agar. Where appropriate streptomycin or kanamycinwere added to growth media at a concentration of 50 μg/ml.

Formaldehyde-treatment of E. coli.

E coli CWG308 or E. coli CWG308:pJCP-Gb3 cells were grown overnight inLB broth supplemented with 20 μg/ml IPTG, and 50 μg/ml kanamycin forCWG308:pJCP-Gb3. Cells were harvested by centrifugation, washed andresuspended in PBS at a density of 10¹⁰ CFU/ml (equivalent to 20 mg dryweight of cells per ml). Formaldehyde was added to a final concentrationof 1% (vol/vol) and the suspension was held at 4□C for 16 hours. Cellswere then washed twice with PBS to remove the formaldehyde andresuspended at the same density in sterile PBS. Complete killing of theE. coli suspensions was confirmed by culture. Suspensions were stored at4° C. for up to two weeks before use.

In vivo protection studies.

The streptomycin-treated mouse model of STEC-induced renal injury hasbeen described previously (Example 1,40,22). Male 5-6 week old Balb/cmice were given oral streptomycin (5 mg/ml in drinking water) for 24hours before oral challenge with 1×10⁸ CFU of the streptomycin-resistantSTEC. Successful colonisation of each mouse, and maintenance at a levelof at least 10⁹ CFU/gm, was confirmed by quantitative culture of faeceson MacConkey agar supplemented with streptomycin. Mice were then givenoral doses of approximately 8 mg dry weight of either CWG308 orCWG308:pJCP-Gb3 (formaldehyde-killed) freshly resuspended in 60 μl of20% sucrose, 10% NaHCO₃, twice or three times daily for up to 12 days.Oral streptomycin was continued throughout the experiment. The survivaltimes of mice in each of the groups were recorded. The differences insurvival rate between STEC-challenged mice treated with killed CWG308 orCWG308:pJCP-Gb3 were analysed using the Fisher exact test. Kidneys werealso removed from selected mice, fixed in formalin andhematoxylin/eosin(HE)-stained sections were examined for histologicalevidence of renal injury.

Results

In an initial experiment we examined the degree of protection againstthe highly virulent STEC strain 97MW1 afforded by oral administration offormaldehyde-killed CWG308:pJCP-Gb3. Four groups of six mice werechallenged with 97MW1 and then treated with either killed CWG308 orCWG308:pJCP-Gb3. The dose administered (approximately 8 mg dry weight)was the same as that used in example 1 for live bacteria, and this wasgiven either twice daily (ie. every 12 hours, as in our previous study)or three times daily (every 8 hours). FIG. 1 shows that allSTEC-challenged mice treated with CWG308 died, with a median survivaltime of approximately four days. Five of the six mice which were treatedwith CWG308:pJCP-Gb3 twice daily survived; all six mice which receivedthree doses per day were alive and well at the termination of theexperiment. For both of these groups, the survival rate wassignificantly better than that of the respective control group treatedwith CWG308 (P<0.005). Histological examination of kidneys removed fromCWG308-treated mice revealed extensive Stx-induced tubular necrosisconsistent with that seen in previous studies (40,22). In contrast,kidneys removed at the end of the experiment from STEC-challenged micetreated with CWG308:pJCP-Gb3 were indistinguishable from those ofunchallenged healthy mice (FIG. 2).

We then investigated the impact of delaying commencement of therapy onsurvival of STEC-challenged mice. Six groups of four mice werechallenged with 97MW1. For five of the groups, therapy withformaldehyde-killed CWG308:pJCP-Gb3 (8 mg dry weight administered8-hourly) was commenced immediately, or after a delay of 8, 16, 24 or 48hours; the sixth group did not receive treatment. As shown in FIG. 3,all mice in the untreated group died within four days. All of the micealso died when treatment with CWG308:pJCP-Gb3 was commenced 24 or 48hours after challenge, although the median survival time was extendedslightly. Only one mouse survived when therapy was commenced after 16hours, but all mice survived when treatment commenced either immediatelyor eight hours after challenge with 97MW1 (P<0.025, compared with theuntreated group). 97MW1 is a highly virulent O113:1H21 STEC strain whichgrows rapidly in the mouse gut and carries three stx2-related genes(39). Thus, it is capable of releasing large amounts of Stx2 into thegut lumen within hours of infection, and this probably contributes tothe rapidly fulminant course of disease. In humans, the lag betweenacquisition of STEC infection and onset of HUS may be as much as twoweeks, and so the above model may underestimate the extent to whichcommencement of treatment can be delayed. Accordingly, we conducted asimilar experiment to that described above using a somewhat lessvirulent STEC challenge strain, 98NK2. This is also a O113:H21 strainand is closely related to 97MW1, on the basis of pulsed-field gelelectrophoretic analysis of genomic DNA, but it carries only one stx2gene (39). Nevertheless, both strains produce similar levels of Stx invitro; the titre in culture lysates (determined by Vero cellcytotoxicity assay) was 8.2×10⁶ tissue culture cytotoxic doses per ml.Using 98NK2 as the challenge strain, five of eight untreated mice died,with a median survival time of six days (FIG. 4). In contrast, all eightmice survived in the groups in which treatment with CWG308:pJCP-Gb3commenced either 0, 8, 16 or 24 hours after challenge (P<0.025). Six ofthe eight mice also survived when treatment commenced 48 hours afterchallenge.

Conclusion

The results of this study unequivocally demonstrate that oraladministration of formaldehyde-killed recombinant bacteria expressing amimic of the Stx receptor protects mice from otherwise fatal challengewith a highly virulent STEC strain. The dose of bacteria used wassimilar to that employed in Example 1. Thus, the capacity to survive inthe gut is not an essential feature of this novel therapeutic agent.However, in order to maintain 100% protection, it was necessary toadminister formaldehyde-killed cells three times rather than twicedaily. The slight reduction in protective efficacy observed with twicedaily administration is probably a consequence of clearance of thetoxin-binding agent between doses. Estimates of the gut transit time formice are of the order of eight hours, and so at lower treatmentfrequencies, mice may be unprotected for the latter portion of eachtreatment period.

Commencement of therapy immediately after challenge was 100% protective,but in the human setting such early intervention will only be possiblefor contacts of confirmed cases, who have not yet, or have only just,become infected with STEC. When using the highly virulent challengestrain 97MW1 in our mouse model, delaying commencement of therapy withformaldehyde-killed CWG309:pJCP-Gb3 by 16 or more hours resulted in lossof protection. However, the window of opportunity for treatment wasextended to 24-48 hours when a less virulent challenge strain (98NK2)was used. 98NK2 is closely related to 97MW1 and has a similar gutcolonisation capacity in the mouse model, as judged by quantitativeculture of faeces (result not presented). The principal differencebetween the two strains is that 97MW1 has three stx2 genes whereas 98NK2has only one. Nevertheless 98NK2 has high human virulence and was thefirst LEE-negative STEC strain to be associated with an outbreak of HUS(39). Moreover, 98NK2 is more virulent in the mouse model than mostO157:H7 STEC strains.

Although the median survival time of unprotected mice challenged with98NK2 was six days, compared with only four days for those challengedwith 97MW1, this still represents a significant time compressionrelative to the kinetics of human disease. In the mouse model,streptomycin treatment eliminates endogenous gut flora prior tochallenge, and the STEC do not have to compete with other organisms.Under these circumstances, the numbers of STEC in the gut increase veryrapidly to 10⁹ to 10¹⁰ CFU per g faeces. Thus, the host is exposed tovery high levels of Stx in the lumen almost from the outset, andpresumably an ultimately lethal dose is absorbed into the circulationrelatively early in the course of infection. In human disease, ingesteddoses of STEC are usually very low and the pathogen must establishcolonisation in competition with endogenous flora. Thus, the time lagbetween actual infection and onset of systemic complications such as HUSis probably of the order of two weeks. In view of these considerations,it seems probable that a significantly broader window will exist fortreatment of human infections. This is supported by the preliminaryfindings of a phase II clinical trial of a synthetic Stx-binding agentSynsorb-Pk for the prevention of progression of STEC disease in childrenfrom diarrhea to HUS. Treatment was associated with a 40% reduction inprogression if commenced within three days of onset of gastrointestinalsymptoms (45). However, the number of patients was small and astatistically significant difference between treatment and placebogroups was not demonstrable. The practical difficulties of conductingsuch efficacy trials are considerable, particularly given the lowincidence of sporadic STEC cases and the unpredictability of outbreaks.The need to target patients in the early stage of illness is alsocomplicated by the inevitable delays associated with laboratoryconfirmation of STEC infection; retrospective exclusion of patientswhose stool samples ultimately prove to be negative for STEC can upsetrandomization.

In Example 1 we demonstrated that the in vitro Stx-binding capacity ofCWG308:pJCP-Gb3 was 10,000 times better than that reported by others forSynsorb-Pk (24,26). For this reason, we would anticipate improved invivo performance in humans relative to Synsorb-Pk, although this canonly be determined in a large scale clinical trial. Another importantconsideration is that the Stx-binding microorganism is likely to beextremely cheap to produce on a large scale, and the formaldehydetreatment should preserve it such that it has a long shelf life,particularly in dried form. Low cost and long shelf life will permitpresumptive treatment of persons with suspected STEC disease, pendingthe results of laboratory analysis of faecal samples. This is animportant consideration, since the findings of this study indicate thatearly commencement of therapy will be essential to prevent progressionof disease to life-threatening systemic complications.

Example 3

Neutralization of Shiga Toxins Stx1, Stx2c and Stx2e by RecombinantBacteria Expressing Mimics of Globotriose and Globotetraose

The construct of example 1, was somewhat less effective at neutralizingthe variant toxin Stx2e produced by STEC strains associated with pigletoedema disease This was not unexpected since Stx2e has been reported tohave a different receptor specificity, recognising globotetraosylceramide (Gb₄; GalNAcβ[1→3]Galα[1→4]Galβ[1→4]Glc-ceramide)preferentially over Gb₃ (17). Piglet oedema disease is a serious,frequently fatal STEC-related illness characterized by neurologicalsymptoms including ataxia, convulsions and paralysis; oedema istypically present in the eyelids, brain, stomach, intestine andmesentery of the colon. It is caused by particular STEC serotypes (mostcommonly O138:K81,O139:K82 and O141:K85), which are not associated withhuman disease (16,46). The altered glycolipid receptor specificityaffects the tissue tropism of the toxin accounting for the distinctiveclinical presentation of oedema disease. Oedema disease occursprincipally at the time of weaning and so incorporation of an effectiveStx2e binding agent into the feed should be capable of preventingdisease outbreaks and the associated economic losses. In this example wehave constructed a recombinant microorganism expressing globotetraose onits surface and examined its capacity to bind and neutralize Stx2e invitro.

Construction of an E. coli CWG308 Derivative ExpressingGalNAcβ[1→3]Galα[1→4]Galβ[1→4]Glc

Globotetraose differs from globotriose only by the additionalN-acetylgalactosamine (GalNAc) linked (1→3) to the terminal galactose(Gal). Thus, insertion of a gene encoding the appropriate GalNActransferase into pJCP-Gb₃ would be expected to direct globotetraoseexpression when introduced into CWG308. The Neisseria lgt locus includessuch a gene (IgtD), but this contains a poly-G tract and so is unstablebecause of susceptibility to slipped strand mispairing (33,34). Toovercome this we mutagenized the lgtD gene by overlap extension PCRusing N. gonorrhoeae chromosomal DNA as template. The 5′ portion of lgtDwas amplified using primers 5′-CAGACGGGATCCGACGTATCGGAAAAGGAGAAAC-3′(LGTDF) (SEQ ID NO:11) incorporating a BamHI site and5′-GCGCGCAATATATTCACCGCCACCCGACTTTGCC-3′ (LGTDOLR) (SEQ ID NO:12). The3′ portion of lgtD was amplified using primers5′-GGCAAAGTCGGGTGGCGGTGAATATATTGCGCGC-3′ (LGTDOLF) (SEQ ID NO:13) and5′-CATGATGGATCCTGTTCGGTTTCAATAGC-3′ (LGTDR) (SEQ ID NO:14) alsoincorporating a BamHI site. PCR was performed using the Expand™ HighFidelity PCR System (Roche Molecular Biochemicals) under conditionsrecommended by the supplier. The two PCR products were then purified,aliquots were mixed, and the complete lgtD coding sequence with thedesired modifications was amplified using primers LGTDF and LGTDR. Thisprocedure mutates GGG codons in the poly-G tract to GGT or GGC (all ofwhich encode Gly), eliminating the risk of slipped-strand mispairingwithout changing the encoded amino acid sequence. The modified PCRproduct was then digested with BamHI and cloned into similarly digestedpJCP-Gb₃ between lgtC and lgtE and transformed into E. coli JM109 (47).Insertion of lgtD with the correct mutations and the appropriateorientation was confirmed by sequence analysis of plasmid DNA usingcustom made oligonucleotide primers and dye-terminator chemistry on anABI model 377 automated DNA sequencer. That part of the lgtD genesequence that has been altered is shown below, the sequence shown istaken from nucleotide 3576 to 3586 of genbank accession number U14554,altered nucleotides are shown in bold and underlined. GGGTGGCGGTG (SEQID NO:15). This plasmid (designated pJCP-IgtCDE) was then transformedinto E. coli CWG308.

LPS was then purified from the above strain as well as from E. coliCWG308 and CWG308:pJCP-Gb3 and analysed by SDS-polyacrylamide gelelectrophoresis with silver-staining as previously described (64).Whilst there was a clear difference in mobility of the LPS from CWG308and CWG308:pJCP-Gb3, expression of the additional transferase gene inCWG308:pJCP-IgtCDE did not further retard LPS mobility (FIG. 5). Thiscould be explained either by failure to produce functional LgtD or byabsence of the essential precursor UDP-GalNAc. This would require afunctional UDP-GalNAc-4-epimerase, an enzyme not necessarily present inall E. coli strains. In a previous study (49) we described the geneticlocus for biosynthesis of E. coli O113 O-antigen, the repeat unitstructure of which includes GalNAc. This locus contains two genes(designated gne and wbnF) encoding proteins with similarity tonucleotide sugar epimerases and we postulated that one or other of thesemay be a functional UDP-GalNAc-4-epimerase. We therefore amplified thegne and wbnF genes from E. coli O113 chromosomal DNA using primers5′-TTTATTAAGCTTCCAATTAAGG AGGTAACTC-3′ (SEQ ID NO:16) and5′-AATTACAAGCTTATAATTTTAATTACCA TACCC-3′ (SEQ ID NO:17) for gne andprimers 5′-ATATTCAAGCTTGAGTGAGGAT TATAAATGAAATT-3′ (SEQ ID NO:17) and5′-TTTCTTAAGCTTTTGTAAAATCAAA CTTTATAGAAG-3′ (SEQ ID NO:18) for wbnF(each primer incorporates a HindIII site). Each PCR product waspurified, digested with HindIII and ligated with HindIII-digestedpJCP-lgtCDE and then transformed into E. coli JM109. Correct insertionand orientation of each construct (designated pJCP-lgtCDE/gne andpJCP-lgtCDE/wbnF) was confirmed by sequence analysis, and then eachplasmid was transformed into CWG308. Comparison of the electrophoreticmobility of LPS purified from these recombinant strains (FIG. 5)indicated that expression of the gne gene resulted in an increase inmolecular size of the LPS. This gene was originally designated galE (15)because it encoded a product with a high degree of similarity toputative GalE proteins (UDP-Glc4-epimerases) from a large number ofbacteria, the most closely related being that from Yersiniaenterocolitica O:8 (57% identity, 73% similarity) (23). However, theYersinia galE gene is now designated gne on the Bacterial PolysaccharideGene Database (available at microbio.usyd.edu.au/BPGD/default.htm) andthe function of its product is listed as a UDP-GalNAc-4-epimerase. Giventhe high degree of similarity between the Yersinia and E. coli O113proteins, and the fact that LgtD is a proven GalNAc transferase (33), weconclude that galE from the E. coli O113 rfb locus also encodes afunctional UDP-GalNAc-4epimerase, and accordingly it has been renamedgne.

Adsorption/neutralization of Stx

The capacity of the above CWG308 derivatives to adsorb and neutralizevarious Stx types was then assessed. Filter-sterilized French pressurecell (FPC) lysates of E. coli JM109:pJCP522 (13) and JM109:pJCP521 (11)were used as a source of Stx1 and Stx2c, respectively. For Stx2e, wefirst PCR-amplified the complete stx2e operon from chromosomal DNAextracted from an O141 STEC strain isolated from a piglet with oedemadisease. The primers used were 5′-GCATCATGCGTTGTTAGCTC-3′ (SEQ ID NO:19)and 5′-AAAGACGCGCATAAATAAACCG-3′ (SEQ ID NO:20). The PCR product waspurified and blunt-cloned into SmaI-digested pBluescript-SK (Stratagene,La Joll, Calif.) and then transformed into E. coli JM109. The insert ofthis plasmid (designated pJCP543) was sequenced and found to beidentical to that previously published for six2e (50), except for asingle nucleotide substitution in the A subunit coding region which didnot affect the amino acid sequence. Accordingly, a FPC lysate ofJM109:pJCP543 was used as a source of Stx2e. The crude Stx extracts wereprepared by growing the various E. coli JM109 derivatives in 10 ml LuriaBertani (LB) broth supplemented with 50 μg/ml ampicillin overnight at37° C. Cells were harvested by centrifugation and resuspended in 10 mlphosphate-buffered saline, pH 7.2 (PBS), and lysed in a French pressurecell (FPC) operated at 12,000 p.s.i. Lysates were then sterilized bypassage through a 0.45 μm filter.

E. coli CWG308, CWG308:pJCP-Gb₃, CWG308:pJCP-lgtCDE,CWG308:pJCP-lgtCDE/gne and CWG308:pJCP-lgtCDE/wbnF were then grownovernight in LB supplemented with 20 μg/ml IPTG and 50 μg/ml kanamycin(except for CWG308). Cells were harvested by centrifugation, washed andresuspended in PBS at a density of 10⁹ CFU/ml. Aliquots (250 μl) of theStx1, Stx2c, and Stx2e extracts were incubated with 500 μl of each ofthe above suspensions, or PBS, for 1 hour at 37° C. with gentleagitation. The mixtures were then centrifuged and the supernatants werefilter-sterilized. Cytotoxicity of the supernatant fraction was thenassayed using Vero (African green monkey kidney) cells, which are highlysusceptible to all Stx-related toxins (2). Twelve serial 2-folddilutions were prepared in tissue culture medium (Dulbecco's ModifiedEagles Medium buffered with 20 mM HEPES, and supplemented with 2 mML-glutamine, 50 IU/ml penicilin and 50 μg/ml streptomycin), commencingat a dilution of 1:1 for Stx2e, or 1:20 for Stx1 or Stx2c. Fifty μl ofeach dilution was transferred onto washed Vero cell monolayers in 96welltissue culture trays, and after 30 min incubation at 37° C., a further150 μl of culture medium was added to each well. Cells were examinedmicroscopically after 72 hours incubation at 37° C., and scored forcytotoxicity. The endpoint Stx titre (cytotoxic doses [CD] per ml) wasdefined as the reciprocal of the highest dilution resulting incytotoxicity in at least 10% of the cells in a given monolayer. As apermanent record, cell monolayers were then fixed in 3.8%formaldehyde-PBS and stained with crystal violet. The percent of Stxadsorbed/neutralized was calculated using the formula 100−(100×CD_(CELLS)/ CD_(PBS)), where CD_(CELLS) is the Stx titre in the extractsincubated with the CWG308 derivatives and CD_(PBS) is the Stx titre inthe respective Stx extract treated only with PBS. As shown in Table 1,CWG308 exhibited no neutralization activity, whereas CWG308:pJCP-Gb₃bound 99.9, 99.2 and 98.4% of the cytotoxicity of Stx1, Stx2c and Stx2e,respectively. This is in accordance with our previous findings for thisglobotriose-expressing construct (Example 1) except for a slightlyimproved neutralization of Stx2e. In Example 1 we observed 87.5%neutralization using a crude lysate of a wild type STEC isolate from acase of oedema disease as a source of Stx2e, but some of the residualcytotoxicity may have been due to the presence of other toxicsubstances. Neutralization of the various toxin types was notsignificantly diminished for CWG308:pJCP-lgtCDE andCWG308:pJCP-lgtCDE/wbnF, which was not surprising given that PAGEanalysis indicated that the LPS from both of these strains wasindistinguishable from that of CWG308:pJCP-Gb₃. However, neutralizationof both Stx1 and Stx2c was significantly lower forCWG308:pJCP-lgtCDE/gne, which is consistent with the alteredelectrophoretic mobility of its LPS. Interestingly, it exhibited thesame in vitro neutralization activity against Stx2e as the otherconstructs (98.4%), in spite of expression of what has hitherto beenbelieved to be the preferred receptor for this toxin type.

Conclusion

In the present study we have modified the globotriose-expressingbacterium CWG308:pJCP-Gb₃ (example 1) such that it expressesglobotetraose (the preferred receptor for Stx2e) by introducingadditional genes encoding a GalNAc transferase (IgtD) and aUDP-GalNAc-4-epimerase (gne). Addition of an extra sugar residue to theouter LPS core required both genes, and was demonstrated byelectrophoretic analysis. Furthermore, the fact that the LPS migrated asa single species implied that this reaction proceeds to completion. Theglobotetraose-expressing bacterium had a reduced capacity to neutralizeStx1 and Stx2c in vitro compared to the globotriose expressing bacteriumpresumably because the terminal GalNAc residue sterically hinders theinteraction between the Stx B subunit and the (now subterminal)globotriose moiety. However, its capacity to bind Stx2e was similar tothat of the globotriose-expressing construct; both neutralized 98.4% ofthe cytotoxicity in lysates of E. coli JM109 expressing cloned sx_(2e).It has long been held that the piglet oedema disease-associated toxinStx2e has a higher affinity for Gb₄ than for Gb₃ (17,13,51). Thus, thefindings of this study were somewhat unexpected. Some of the earlystudies on Stx receptor specificity involved overlaying glycolipidsseparated by thin layer chromatography with toxin. However, it has beensuggested that the polyisobutylmethacrylate used in these studies (tostabilize the silica gel prior to reaction of the separated lipids withtoxin) may have induced conformational changes in the carbohydratemoieties which affected toxin-receptor interactions (52). Receptorspecificity was also examined on the basis of susceptibility of celllines containing varying amounts of Gb₃ and Gb₄ to the toxin.Interestingly, fatty acyl chain length is known to influence theinteraction of Gb₃ with Stx1 and Stx2 to differing extents, and so it ispossible that factors other than the structure of the oligosaccharidecomponent may have been a compounding factor in the cell culture studies(13). In the present study the globotriose and globotetraose moietieswere expressed on an otherwise identical platform comprising the innercore oligosaccharide and the lipid A components of E. coli LPS. Thus,differences in toxin-receptor interactions (or lack thereof) trulyreflect the impact of oligosaccharide structure and conformation.

Example 4

Clostridium difficile

C. difficile infection is associated with broad spectrum antibiotictherapy and is the commonest cause of infectious diarrhoea andlife-threatening pseudomembranous colitis in hospitalized patients.Antibiotic therapy permits overgrowth of the gut by this bacterium,which elaborates two potent cytotoxins (exotoxins A and B). Exotoxin Ais enterotoxic and is essential for human virulence; exotoxin B can onlydamage host tissues after destruction of the epithelial barrier byexotoxin A (53). C. difficile exotoxin A binds to several humanglycolipids, all of which contain Galβ[1→4]GlcNAc moiety. Genes encodingtransferases capable of assembling this epitope are also found in theNeisseria lgt locus. Expression of lgtABE in E. coli CWG308 waspredicted to result in synthesis of a LPS outer core oligosaccharidecomprising Galβ[1→4]GlcNAcβ[1→3] Galβ[1→4]Glc→(lacto-N-neotetraose). ThelgtA-B genes were amplified from N. gonorrhoeae DNA by PCR, and thepoly-G tract in lgtA was mutagenized by overlap-extension PCR, asfollows. The 5′ portion of lgtA was amplified using primers5′-CAGGCGAATTCAAATTATCGGGAGAGTA-3′ (LGTAF) (SEQ ID NO:21) incorporatingan EcoRI site (underlined) and5′-ATATTCGCCACCGCCACCGCCCGACTTTGCCAATTCG-3′ (LGTAOLR) (SEQ ID NO:22).The 3′ portion of lgtA and all of lgtB was amplified using primers5′-GTCGGGCGGTGGCGGT GGCGAATATATTGCGCGCACCG-3′ (LGTAOLF) (SEQ ID NO:23)and 5′-CATCTTGGATCCTTTTATTGGAAAGGCAC-3′ (LGTBR) (SEQ ID NO:24)incorporating a BamHI site (underlined). PCR was performed using theExpand™ High Fidelity PCR System (Roche Molecular Biochemicals) underconditions recommended by the supplier. The two PCR products were thenpurified, aliquots were mixed, and the complete lgtAB coding sequencewith the desired modifications was amplified using primers LGTAF andLGTBR. This procedure mutates the four consecutive GGG codons in thepoly-G tract in lgtA to GGT or GGC (all of which encode Gly),eliminating the risk of slipped-strand mispairing without changing theencoded amino acid sequence. That part of the lgtA gene sequence thathas been altered is shown below, the sequence shown is taken fromnucleotide 699 to 715 of GenBank accession number U14554, alterednucleotides are shown in bold and underlined. GGGCGGTGGCGGTGGCG (SEQ IDNO:25). The modified PCR product was then digested with EcoRI and BamHIand cloned into the similarly digested derivative of pK184 containinglgtE described earlier. This places lgtAB between the pK184 vectorpromoter and lgtE, with all three genes in the same orientation suchthat they will be co-transcribed. Insertion of lgtAB with the correctmutations and the appropriate orientation was confirmed by sequenceanalysis of plasmid DNA using custom made oligonucleotide primers anddye-terminator chemistry on an ABI model 377 automated DNA sequencer.This plasmid (designated pJCP-LNT) was then transformed into E. coliCWG308.

LPS was then purified from CWG308 derivatives expressing this constructand analysed by SDS-PAGE (using silver staining). This confirmed thatexpression of lgtABE (encoded on pJCP-LNT) in E. coli CWG308 resulted inproduction of a higher molecular weight LPS than either CWG308 or CWG308carrying pJCP-Gb₃. This is consistent with expression of theGalβ[1→4]GlcNAcβ[1→3]Galβ[1→4]Glc→(lacto-N-neotetraose) epitope on thecell surface. Capacity to bind and neutralize exotoxin A can then beassessed using filter-sterilized C. difficile culture supernatant orlysate of E. coli K12 expressing cloned toxin genes or purified toxin Aas a source of toxin. Such extracts will be incubated with suspensionsof (live or formalin-killed) CWG308:pJCP-LNT (or control constructs)with gentle mixing for 1 h at 37° C. Cells can be removed bycentrifugation, and serial 2-fold dilutions of filter-sterilizedsupernatant will be transferred to Vero cell monolayers (in 96-welltrays). Monolayers will be scored for C. difficile cytotoxicity at 24 hand the endpoint titre determined. The endpoint titre (cytotoxic doses[CD] per ml) is defined as the reciprocal of the highest dilutionresulting in cytotoxicity (characteristic rounding up of cells) in atleast 10% of a given monolayer. As a permanent record, cell monolayerscan be fixed in 3.8% formaldehyde-PBS and stained with crystal violet.The percent of toxin neutralized can be calculated using the formula100−(100×CD_(CELLS)/ CD_(PBS)), where CD_(CELLS) is the toxin titre inthe extracts incubated with a given CWG308 construct, and CD_(PBS) isthe toxin titre in the respective sample treated only with PBS.

Interestingly, in vitro studies indicate that even stronger bindingoccurs between exotoxin A and the trisaccharideGalα[1→3]Galβ[1→4]GlcNAc→, even though it is not present in humans (54).A strain expressing this epitope can be constructed by incorporating agene encoding a transferase capable of forming the necessaryGalα[1→3]Galβ linkage into pJCP-LNT. Databases will be searched for asource of such a transferase. An alternative source is the capsule locusof Streptococcus pneumoniae type 11F, which is known to encode such anenzyme. Candidate transferase genes from this locus can be amplified byPCR and cloned into the lacto-N-neotetraose-encoding construct. Heerzeet al, (U.S. Pat. No. 5,635,606) have also demonstrated that severalother immobilized oligosaccharides, including the trisaccharideGalα[1→3]Galβ[1→4]Glc- are capable of binding C. difficile toxin A. Astrain expressing this latter epitope in its outer core LPS can beconstructed by expressing the Galα[1→3]Galβ transferase gene referred toabove along with just lgtE in E. coli CWG308. These derivatives can betested for toxin binding in parallel with the above constructs.

Although C. difficile toxin A is necessary for virulence, significanttissue damage may also result from the action of toxin B, particularlyif adsorption of toxin A is incomplete. The actual human receptor fortoxin B has not been characterized, but Heerze et al. (U.S. Pat. No.6,013,635) disclose that immobilized Glcα[1→6]Glc- (isomaltose) orGlcα[1→6]Glcα[1→6]Glc→(isomaltotriose) bind and neutralize toxin B invitro. Thus, expression of an appropriate glucosyl transferase gene(s)in CWG308, is predicted to confer the capacity to neutralize thiscytotoxin.

Example 5

A Receptor Mimic Construct for Treatment of Traveller's Diarrhoea

Traveller's diarrhoea is caused by infection with enterotoxigenic E.coli (ETEC). Heat labile enterotoxin (LT) is a major ETEC virulencefactor. It binds principally to the ganglioside G_(Ml)(Galβ[1→3]GalNACβ[1→4](NeuAcα[2→3]) Galβ[1→4]Glcβ→ceramide) on theenterocyte surface and induces diarrhoea by stimulating adenylatecyclase activity (55). LT is also capable of binding to a variety ofother glycoconjugates, as described by Heerze et al. (U.S. Pat. No.6,069,137). The principal adhesins of ETEC are a family of pili calledcolonization factor antigens (CFA) or coli surface antigens (CS), andthese enable the ETEC to colonize the small intestine, an essential stepof the pathogenic process. Several potential oligosaccharide receptorshave been identified for CFAs, including asialo G_(M1)(Galβ[1→3]GalNAcβ[1→4]Galβ[1→4]Glcβ→ceramide) as well as several sialicacid containing glycoconjugates (56,57). In addition, the disaccharideGalNAc[1→4]Gal has been shown to be a binding sequence for ETEC thatexpress CS3 pili (58). Thus, administration of appropriate receptormimic constructs would be expected to neutralize the enterotoxic effectsof LT as well as blockade adherence of ETEC to the intestinal epitheliumImportantly, LT is structurally and functionally related to choleratoxin (CT) produced by Vibrio cholerae, which also binds to G_(M1).Thus, any product developed for neutralization of LT and treatment oftraveller's diarrhoea will also be effective against cholera.

GM1 is mimicked by the LPS outer core of several Campylobacter jejunistrains including the strain deposited as NCTC 11168. These are known tobe capable of binding purified CT in vitro (59). The genome sequence ofNCTC 11168 is available at sanger.ac.uk/Projects/C jejuni. Moreover,Linton et al. (60) have identified the LOS-encoding region within thissequence and have functionally characterized one of the genes (wlaN)involved in synthesis of the G_(M1) mimic LOS structure. As with some ofthe Neisseria lgt genes described previously, wlaN has a poly-G tractwhich will have to be mutagenized to stabilize expression, as describedabove for lgtA, lgtC, and lgtD. Sequence data for the C. jejuni LOSregion will be used to design primers for PCR amplification of theappropriate genes for assembly of the G_(M1) mimic on the outer core LPSof CWG308. Additional CWG308 derivatives expressing the alternativeLT-binding oligosaccharide epitopes listed in U.S. Pat. No. 6,069,137can also be constructed. LPS can be extracted from each construct andanalysed by SDS-PAGE as before. CWG308 derivatives can then be testedfor binding and neutralization of purified CT and LT using a commercialELISA assay or by direct blotting using commercially availableperoxidase-conjugated CT. Protective capacity of any toxin-bindingconstructs can also be tested in an infant mouse cholera model.

CWG308 derivatives capable of interfering with adherence of ETEC toepithelial cells can be constructed by deletion of the sialyltransferase gene from the G_(M1)-expressing strain. Derivativesexpressing alternative oligosaccharide receptor mimics capable ofblocking adherence of a pathogen can also be constructed. Where data onthe receptor specificity of a given CFA/CS family are not available,attempts can be made to determine this by competitive adherenceinhibition assay using a panel of soluble mono-, di- andtri-saccharides. This will enable selection of appropriate transferasegenes to direct assembly of the chosen epitope on the CWG308 surface.The various constructs can then be tested for capacity to agglutinate avariety of ETEC strains representing all common CFA/CS families, and forcapacity to blockade in vitro adherence of these ETEC strains to HEp-2and Henle 407 cells.

Example 6

Uropathogenic E. coli and Porcine Enterotoxigenic E. coli

Two further model systems can be used to demonstrate the utility ofrecombinant receptor mimic technology for blockade of bacterialadhesion. The first is adherence of uropathogenic E. coli ; this ismediated by Pap pili, which are known to target the terminalGalα[1→4]Galβ moiety of Gb₃ receptors on the uroepithelium (54).Accordingly it is proposed to test the capacity of our existingCWG308:pJCP-Gb₃ construct of Example 1 to agglutinate referenceuropathogenic E. coil strains as well as E. coli K-12 clones expressingcloned pup genes. Capacity to blockade in vitro adherence toGb₃-expressing epithelial cell lines (HeLa or Vero cells) can also beexamined using standard tissue culture assays. The second example isadhesion of porcine enterotoxigenic E. coli strains which express K88adfimbriae. These pili are known to be specific for facto-N-neotretraose(43), and so CWG308:pJCP-LNT constructed as described above for use withC. difficile is predicted to be capable of binding to K88ad fimbriae.

Example 7

Entamoeba histolyticum

E. histilyticum is an enteric protozoan parasite which causes nearly 40million cases of colitis or liver abscess each year, of which about100,000 are fatal. Pathogenesis involves adherence to colonic mucin andcolonic epithelial cells, followed by mucosal invasion and hostcytolysis (61). Adherence is mediated by a Gal/GalNAc specific lectin,and inhibition of this lectin activity blocks cytotoxicity. E.histolyticum binds monomeric and polymeric GalNAc-BSA conjugates withvery high efficiency (61). Thus E. coli derivatives expressing GalNAcmoieties on their surface are proposed to be used for the treatment andprevention of E. histolyticum infections. In the first instance, we caninvestigate whether the CWG308:Gb₄ receptor mimic construct of Example 3(which has a terminal GalNAc moiety) is capable of blockading theinfectivity of E. histolyticum trophozoites. This can be assayed invitro by binding to CHO cells, and by cytopathic effect for Caco-2 cells(61). Binding by constructs expressing additional GalNAc moieties can beexamined using constructs expressing additional GalNAc transferasegenes.

Example 8

Rotavirus

Although the receptor for human rotavirus is yet to be characterized,that for porcine strains has recently been shown to be the sialatedgangliosides NeuGc-GM₃ and NeuNAc-GM₃. Moreover, similar glycolipidspresent in human breast milk are believed to be responsible for theprotection from rotavirus observed in breast-fed infants.

A sialic acid-containing glycolipid termed lactadhesin has been shown toblock interaction of human rotavirus with its receptor (65), and thussialic acid is proposed to act as a mimic for human rotavirus and may beused for therapeutic purposes.

It will be understood that recombinant microorganisms that form thechimeric carbohydrate receptor mimic can be used in the treatment ofdisease, so that post onset administration can prevent persistence byproviding competition with binding receptors or by inhibiting thebinding of toxin. Equally it will be apparent in circumstances such asin the outbreak of an infectious event that individuals at high ormoderate risk can have the receptor mimics administered to preventinfection.

It will however also be understood that, especially in commercial orsporting animals, administration of the present invention can beprescribed to prevent the onset of a condition. Thus the onset of scoursor oedema disease in weanling pigs can be prevented by addition of themicroorganisms, or extracts from the microorganisms that include theoligosaccharide-containing binding moieties, to feed. This can beadministered over a continuous period during which the weanling pigletsare fed or perhaps at intervals during that period to reduce theseverity or frequency of onset.

Example 9

Detection of Pathogenic Organisms and Toxins Produced by Them

Detection methods, particularly immunological methods require sufficientavidity between the interaction between for example the antibody on theone part and the antigen on the other. It has been found that for thedetection of at least some toxigenic pathogens that monoclonal antibodyreagents have not been sufficiently avid to allow the commercialisationof so called dip stick methods. One of the findings of the presentinvention is the very high capacity of the receptor mimics to interactwith toxins. It is thus probably that the chimeric carbohydrates willnot only be useful as means of inhibiting the affects of infection ortoxin action but also as reagents for the detection of such infectiousagents and toxins.

Thus for example lipopolysaccharide is known as a reagent for coatingbeads, or plastics trays such as ELISA trays, or suitable supportsurfaces. Thus chimeric carbohydrates can be partially or whollypurified from the recombinant microorganism of the second aspect of theinvention and bonded to the support. The sample is then added, washedand then perhaps a suitable labelled antibody used to detect for thepresence of bound toxin.

The following protocol can be used:

LPS-Gb₃ is prepared using the method of Darveau and Hancock (48). LPS isalkali treated to improve solubility and adhesion to plastics. LPS isused to coat ELISA trays. Non-specific binding sites are blocked withBSA. Diluted samples containing toxin are added to wells of the tray.After incubation, the wells are washed, then incubated with anti-toxinmonoclonal antibody. After incubation, the well are washed, then anenzyme conjugated secondary antibody is added to detected boundanti-toxin antibody. After incubation, the walls are washed, then acolour substrate is added, and developed

An ELISA reader is used to quantitate the level of toxin binding. Thisprotocol is more particularly set out in reference (65).

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It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference for allpurposes. Also, a reference within this specification to a document isnot to be taken as an admission that the disclosure therein constitutescommon general knowledge in Australia.

What is claimed is:
 1. A recombinant bacterium selected from the groupof species consisting of Escherichia coli and Salmonella enterica thatdisplays on its surface a binding moiety that acts as a receptor mimic,the binding moiety being a receptor mimic of a receptor for a toxin of apathogenic microorganism or an adhesin of a pathogenic microorganism,wherein the binding moiety consists of an oligosaccharide whichcomprises a sugar residue that is attached to an acceptor moiety by aglycosyltransferase that is encoded by an exogenous nucleic acid whichis present in the bacterium, said oligosaccharide forming part of alipopolysaccharide molecule.
 2. The recombinant bacterium of claim 1,wherein the oligosaccharide further comprises at least a second sugarresidue that is attached to an acceptor moiety by at least a secondglycosyltransferase, the second glycosyltransferase being encoded by asecond exogenous nucleic acid which is present in the bacterium.
 3. Therecombinant bacterium of claim 1, wherein the toxin is an enterotoxin.4. The recombinant bacterium of claim 1, wherein the toxin is selectedfrom the group consisting of shiga toxins, clostridial toxins, choleratoxins, E. coli enterotoxins, and Staphylococcal enterotoxins.
 5. Therecombinant bacterium of claim 4, wherein the toxin is selected from thegroup consisting of cholera toxin, E. coli heat labile enterotoxin typesI and II, and ST toxins.
 6. The recombinant bacterium of claim 1,wherein the binding moiety comprises an oligosaccharide selected fromthe group consisting of Galα[1→4]Galβ[1→4]Glc, Galα[1→4]Galβ,GalNAcβ[1→3]Galα[1→4]Galβ[1→4]Glc, Galβ[1→4]GlcNAc,Galα[→3]Galβ[1→4]Glc, Galα[1→3]Galβ[1→4GlcNAc,Galβ[1→4]GlcNAcβ[1→3]Galβ[1→4]Glc, Glcα[1→6]Glc, Glcα[1→6]Glcα[1→6]Glc,NeuNAc,

 Galβ[1→3]GalNAcβ[1→4]Galβ[1→4]Glc, GalNAcβ[1→4]Gal, GalNAc, Gal,NeuGc→GM3, and NeuNAc→GM3.
 7. The recombinant bacterium of claim 6,wherein the binding moiety comprises NeuNAc.
 8. The recombinantbacterium of claim 6, wherein the binding moiety comprises theoligosaccharide:


9. The recombinant bacterium of claim 1, wherein the binding moiety is amimic of a natural receptor for adhesins or toxins produced by amicro-organism selected from a group of genera consisting ofEscherichia, Salmonella, Shigella, Citrobacter, Helicobacter, Yersinia,Vibrio, Aeromonas, Campylobacter, Pseudomonas, Pasteurella, Neisseria,Haemophilus, Klebsiella, Staphylococcus, Streptococcus, Clostridium,rotavirus, and Entamoeba.
 10. The recombinant bacterium of claim 1,wherein the bacterium further comprises one or more exogenous enzymesinvolved in synthesis of a nucleotide sugar which serves as a donor forthe glycosyltransferase.
 11. The recombinant bacterium of claim 10,wherein the nucleotide sugar is selected from the group consisting ofGDP-Man, UDP-Glc, UDP-Gal, UDP-GlcNAc, UDP-GalNAc, CMP-sialic acid,GDP-Fuc, and UDP-xylose.
 12. The recombinant bacterium of claim 10,wherein the enzyme is a nucleotide sugar synthetase.
 13. The recombinantbacterium of claim 10, wherein the enzyme is involved in synthesis of anucleotide that comprises the nucleotide sugar.
 14. The recombinantbacterium of claim 10, wherein the enzyme is involved in synthesis of asugar that comprises the nucleotide sugar.
 15. The recombinant bacteriumof claim 10, wherein the one or more sugars transferred to the acceptormolecule by the exogenous glycosyltransferases make up the entirety ofthe receptor mimic.
 16. The recombinant bacterium as in claim 1, whereina combination of sugars of the acceptor molecule and the one or moresugars transferred to the acceptor molecule by the exogenousglycosyltransferases make up the entirety of the receptor mimic.
 17. Therecombinant bacterium as in claim 1, wherein the acceptor moiety is allor a portion of the core of the lipopolysaccharide.
 18. A pharmaceuticalpreparation for enteral administration, said preparation comprising arecombinant delivery bacterium and a pharmaceutically acceptableexcipient, wherein the delivery bacterium is selected from the group ofspecies consisting of Escherichia coli and Salmonella enterica, whereinthe delivery bacterium expresses one or more exogenousglycosyltransferases encoded by an exogenous nucleic acid and anacceptor molecule, wherein said one or more exogenousglycosyltransferases are specific for the transfer of one or more sugarresidues represented progressively from a non reducing terminal end of areceptor of either a toxin of a pathogenic microorganism or an adhesinof a pathogenic microorganism, and further wherein the one or moreglycosyltransferases progressively transfer said one or more sugarresidues onto the acceptor molecule to thereby form a chimericcarbohydrate molecule with an exposed receptor mimic, wherein saidexposed receptor mimic is capable of binding the toxin or the adhesin,and further wherein a combination of sugars of the acceptor molecule andthe one or more sugars transferred to the acceptor molecule make up theentirety of the receptor mimic, and wherein said chimeric carbohydratemolecule is a lipopolysaccharide molecule.
 19. The pharmaceuticalpreparation as in claim 18, wherein the receptor mimic is a mimic of thereceptor of a bacterial toxin.
 20. The pharmaceutical preparation as inclaim 19, wherein the toxin is selected from the group consisting ofshiga toxins, clostridial toxins, cholera toxins, E. coli enterotoxins,and Staphylococcal enterotoxins.
 21. The pharmaceutical preparation asin claim 20, wherein the toxin is a clostridial toxin.
 22. Thepharmaceutical preparation as in claim 18, wherein the receptor mimic ispartially or wholly formed within a sugar moiety selected from the groupconsisting of: Galα[1→4]Galβ[1→4]Glc, Galα[1→4]Galβ,GalNAcβ[1→3]Galα[1→4]Galβ[1→4]Glc, Galβ[1→4]GlcNAc,Galα[1→3]Galβ[1→4]Glc, Galα[1→3]Galβ[1→4]GlcNAc,Galβ[1→4]GlcNAcβ[1→3]Galβ[1→4]Glc, Glcα[1→6]Glc, Glcα[1→6]Glcα[1→6]Glc,NeuNAc,

 Galβ[1→3]GalNAcβ[1→4]Galβ[1→4]Glc, GalNAcβ[1→4]Gal, GalNAc, Gal,NeuGc→GM3, and NeuNAc→GM3.
 23. The pharmaceutical preparation as inclaim 18, wherein one or more exogenous nucleotide sugar precursorsynthesizing enzymes encoded by a second exogenous nucleic acid are alsoexpressed by said delivery bacterium, said sugar precursor enzymesforming precursors to make up said chimeric carbohydrate.
 24. Thepharmaceutical preparation as in claim 18, wherein the deliverybacterium is non harmful and live.
 25. The pharmaceutical preparation asin claim 18, wherein the delivery bacterium is protected by a protectivecapsule or held within a protective matrix.
 26. The pharmaceuticalpreparation as in claim 18, wherein the delivery bacterium is killedbefore administration of the pharmaceutical preparation.
 27. Thepharmaceutical preparation as in claim 26, wherein the deliverybacterium is killed by treatment with a chemical agent selected from thegroup consisting of formalin or thiomersal, or by treatment with abactericidal antibiotic, or by exposure to heat or UV irradiation.
 28. Arecombinant E. coli that displays on its surface a binding moiety thatacts as a receptor mimic when administered to an animal, and competeswith a ligand for binding to a receptor for the ligand, wherein thereceptor mimic consists of an oligosaccharide which comprises a sugarresidue that is attached to an acceptor moiety by a glycosyltransferasethat is encoded by an exogenous nucleic acid which is present in the E.coli, said oligosaccharide forming part of a lipopolysaccharidemolecule.
 29. The recombinant E. coli of claim 28, wherein theoligosaccharide is Galα[1→4]Galβ[1→4]Glc.
 30. The recombinant E. coli ofclaim 28, wherein the oligosaccharide isGalNAcβ[1→3]Galα[1→4]Galβ[1→4]Glc.
 31. The recombinant bacterium as inclaim 1 wherein said bacterium is Escherichia coli.
 32. Thepharmaceutical preparation as in claim 18 wherein said deliverybacterium is Escherichia coli.